Standard Toxicity Tests Research Articles

Integrating the lethal and sublethal effects of toxic compounds into the population dynamics of Daphnia magna: A combination of the DEBtox and matrix population models

Bioassays can be used in aquatic toxicology to provide individual determinations, however ecotoxicology is now attempting to assess the impact of pollution on populations. The main issue is how to infer the impact on an entire population of the toxic effects observed in individuals. At the individual level, Dynamic Energy Budget in Toxicology (DEBtox) theory provides a set of mechanistic models of survival, reproduction and growth continuously as a function of time and exposure concentration, in which the parameters used have clear biological meanings. These models have been designed for the analysis of data provided by the internationally standardized toxicity tests, and sometimes make it clear which is the mode of action of the contaminant. Matrix population models directly yield the population growth rate, the most robust endpoint in risk assessment at the population level. By combining DEBtox theory and matrix population models, we extrapolate every effects of the toxic compound on the individual (reduced fecundity, growth and survival) to the population level. Both lethal and sublethal effects are integrated into a single parameter, the population growth rate, which is calculated continuously against exposure concentration. We can thus compare the consequences at the population level of choosing one or another assumption about the mode of action of the contaminant at the individual level. We used various complementary matrix population models, in order to perform a complete sensitivity analysis, highlighting critical demographic parameters in the evolution of population growth rate as a function of contaminant concentration. Here, we apply this method to a test organism commonly used in ecotoxicology, Daphnia magna, through a case study of cadmium contamination.

Hydrobia ulvae feeding rates: A novel way to assess sediment toxicity

Standard acute toxicity tests are widely used to assess contaminated sediments. However, such tests last 10 d or more and only provide information regarding lethality. Here, we present data concerning the use of a 28-d growth test and a 24-h test using feeding rate, as measured by egestion rate, of the marine snail Hydrobia ulvae. The test was used to assess the toxicity of estuarine sediments from a gradient of heavy metal contamination, and its sensitivity and ease of use were compared with those of 10-d tests using the amphipod crustacean Corophium volutator. Mortality of C. volutator and H. ulvae in 10-d lethal toxicity tests showed similar patterns of sensitivity. Lethality tests with both species showed no effects when carried out using sediments from a number of sites at which ecological impacts are known to occur. By contrast, growth over 28 d in H. ulvae was reduced at all sites where other studies have detected adverse ecological effects. Feeding rate after 24 h also was decreased at moderately contaminated sites where sediments were not acutely toxic, and it was a very good predictor of 28-d growth (r2 = 0.74). Both tests were straightforward to carry out, so H. ulvae has considerable potential as a test organism for chronic toxicity.

Development and application of oxygen production rate assessment to uptake of fluorinated organics by Lemna minor

The effects of contaminant concentration on contaminant removal by Lemna minor were examined under elevated plant densities typical of full surface coverage. Uptake of 3-fluorophenol and 3-trifluoromethylphenol by L. minor was determined at concentrations from 10 to 1750 microM. Because standardized toxicity tests typically do not measure plant activity of L. minor at high plant densities, an oxygen production rate (OP) assessment was developed with consideration of experimental parameters that may inhibit OP by L. minor, including concentrations of carbonate and phosphate buffer in the media, time of assessment, and mass of L. minor. The OP by L. minor was inhibited by increasing media pH and mass of L. minor. The developed assessment used 0.5 g of L. minor per 60 ml of modified Standard Methods medium. A total of 30 h was needed to determine toxicity of fluorinated phenols, with an exposure period of 24 h, an incubation time of 6 h, and negligible analysis time. At concentrations from 10 to 1750 microM, 3-trifluoromethylphenol exhibited a sigmoidal toxicity response. However, 3-fluorophenol was neither toxic nor inhibitory at the experimental concentrations tested. Substantial decreases in uptake rate constants were observed for increasing concentrations of both contaminants, indicating that concentration may be a more important indicator of uptake by L. minor compared with decreasing plant activity because of toxicity.

Determining stressor presence in streams receiving urban and agricultural runoff: Development of a benthic in situ toxicity identification evaluation method

Determining toxicity in streams during storm-water runoff can be highly problematic because of the fluctuating exposures of a multitude of stressors and the difficulty of linking these dynamic exposures with biological effects. An underlying problem with assessing storm-water quality is determining if toxicity exists and then which contaminant is causing the toxicity. The goal of this research is to provide an alternative to standard toxicity testing methods by incorporating an in situ toxicity identification evaluation (TIE) approach. A benthic in situ TIE bioassay (BiTIE) was developed for separating key chemical classes of stressors in streams during both low- and high-flow events to help discern between point and nonpoint sources of pollution. This BiTIE method allows for chemical class fractionation through the use of resins, and these resins are relatively specific for removing nonpolar organics (Dowex Optipore), ammonia (zeolite), and polywool (control). Three indigenous aquatic insects, a mayfly (Isonychia spp.), a caddisfly (Hydropsyche spp.), and a water beetle (Psephenus herricki), were placed in BiTIE chambers that were filled with natural substrates. Acute 96-h exposures were conducted at Honey Creek, New Carlisle, Ohio, USA (reference site), and Little Beavercreek, Beavercreek, Ohio, USA (impaired site). At both sites, significant (p < 0.025) stressor responses were observed using multiple species with polywool or no resin (control) treatments exhibiting < 80% survival and resin treatments with >80% survival. The BiTIE method showed stressor-response relationships in both runoff and base flow events during 96-h exposures. The method appears useful for discerning stressors with indigenous species in situ.

Toxicological assessment of industrial solvents using human cell bioassays: assessment of short-term cytotoxicity and long-term genotoxicity potential

There is an increasing demand for simple toxicological screening methods to assess the human health risk associated with exposure to environmental toxicants. Such screening tools should allow for risk evaluation in terms of both short-term/acute toxicity and longer-term genetic damage, which may lead to mutagenicity and carcinogenicity. We employed a battery of human cell bioassays using the human hepatoma cell-line, HepG2, to assess the cytotoxic and genotoxic potential of environmental pollutants. Here, we demonstrate direct application of these human cell bioassays to the toxicological assessment of a number of industrial solvents that are in common use worldwide. HepG2 cells were exposed to various solvents at concentrations ranging from 25 to 500 ppm. The cells were then analysed using specific protocols for four different adverse effects: cell death/acute cytotoxicity using a neutral red uptake assay, altered enzyme function (often an indicator of cell stress) using the ethoxyresorufin O-deethylase (EROD) bioassay, DNA single strand breaks (SSB), and DNA repair induction, which evaluates mutagenic activity. Using the positive controls, linear dose-response curves were achieved for all four bioassays. The high sensitivity of the tests allowed for environmentally meaningful assessments, and precision studies showed excellent reproducibility for all four bioassays. Comparing the results of the four bioassays on each of 16 industrial solvents allowed for ranking of the anticipated relative human toxicity of these solvents, which were comparable with data from standard toxicity tests and human occupational data. Overall, the study clearly supports the application of the HepG2 cell bioassay system for rapid toxicological screening of many candidate toxicants for both short- and long-term toxicity potential.

Relating chronic toxicity responses to population-level effects: A comparison of population-level parameters for three salmon species as a function of low-level toxicity

Standard laboratory toxicity tests assess the physiological responses of individual organisms to exposure to toxic substances under controlled conditions. Time and space restrictions often prevent the assessment of population-level responses to a toxic substance. Contaminants can affect various biological functions (e.g. growth, fecundity or behavior), which may alter different demographic traits, leading to population-level impacts. In this study, immune suppression, reproductive dysfunction and somatic growth impairment were examined using life history matrix models for coho salmon ( Oncorhynchus kisutch), sockeye salmon ( Oncorhynchus nerka) and chinook salmon ( Oncorhynchus tshawytscha). Our intent was to gauge the relative magnitude of response to toxic effects among species and between life history stages, not provide a specific estimate of population growth rate or abundance. Effects due to immune suppression were modeled as reductions in age-specific survival. Toxic impacts on reproductive function were modeled as a 10% reduction in reproductive contribution for all reproductively mature age groups. Model runs that examined the effect of somatic growth reduction on population parameters incorporated both survival and reproductive impacts. All impacts were modeled as 10% reductions in the affected population demographic parameters. First-year survival and reproductive impacts produced similar population growth rates ( λ), but resulted in different sensitivity and stable age distributions. Modeled somatic growth reduction produced additive effects on survival and reproduction. Toxic stressors producing similar changes in λ did not necessarily produce similar changes in the age distributions. Sensitivity and elasticity analyses demonstrated that changes to the first-year survival rate produced the greatest per-unit effect on λ for each species. Alteration in abundance of mature females also strongly influenced λ. Differences observed between species showed that the number of reproductive ages and time to reproductive maturity were important components for population-level responses. These results emphasize the importance of linking toxicity responses at low concentrations to the demographic traits they affect, and help to highlight the toxicity tests that are more suitable for assessing impacts on the focal species. Additionally, life history modeling is a useful tool for developing testable hypotheses regarding impacts on specific populations as well as for conducting comparisons between populations.

Immunotoxicogenomics: The Potential of Genomics Technology in the Immunotoxicity Risk Assessment Process

Evaluation of xenobiotic-induced changes in gene expression as a method to identify and classify potential toxicants is being pursued by industry and regulatory agencies worldwide. A workshop was held at the Research Triangle Park campus of the Environmental Protection Agency to discuss the current state-of-the-science of "immunotoxicogenomics" and to explore the potential role of genomics techniques for immunotoxicity testing. The genesis of the workshop was the current lack of widely accepted triggering criteria for Tier 1 immunotoxicity testing in the context of routine toxicity testing data, the realization that traditional screening methods would require an inordinate number of animals and are inadequate to handle the number of chemicals that may need to be screened (e.g., high production volume compounds) and the absence of an organized effort to address the state-of-the-science of toxicogenomics in the identification of immunotoxic compounds. The major focus of the meeting was on the theoretical and practical utility of genomics techniques to (1) replace or supplement current immunotoxicity screening procedures, (2) provide insight into potential modes or mechanisms of action, and (3) provide data suitable for immunotoxicity hazard identification or risk assessment. The latter goal is of considerable interest to a variety of stakeholders as a means to reduce animal use and to decrease the cost of conducting and interpreting standard toxicity tests. A number of data gaps were identified that included a lack of dose response and kinetic data for known immunotoxic compounds and a general lack of data correlating genomic alterations to functional changes observed in vivo. Participants concluded that a genomics approach to screen chemicals for immunotoxic potential or to generate data useful to risk assessors holds promise but that routine use of these methods is years in the future. However, recent progress in molecular immunology has made mode and mechanism of action studies much more practical. Furthermore, a variety of published immunotoxicity studies suggest that microarray analysis is already a practical means to explore pathway-level changes that lead to altered immune function. To help move the science of immunotoxicogenomics forward, a partnership of industry, academia, and government was suggested to address data gaps, validation, quality assurance, and protocol development.

Low Levels of Environmental Ammonia Increase Susceptibility to Disease in Chinook Salmon Smolts

Ammonia criteria are established using data from standardized toxicity tests involving healthy animals. Both intrinsic and extrinsic environmental changes affect the immune system, but few toxicity studies consider the overall impact on this system and potential changes in resistance to infection. To investigate the effects of subacute levels of ammonia in coastal waters on physiological and immunological systems of fish, juvenile Chinook salmon were maintained in seawater (10 degrees C, pH 7.8) and exposed to two concentrations of ammonia, 2.5 and 10 mg/L total nitrogen. Both test levels resulted in increased internal levels of ammonia in the fish. Neither treatment level affected feeding rates. Over a time course of 10 d, numerous significant effects were observed. White blood cell counts changed significantly, as did respiratory burst activity, plasma lysozyme activity, and plasma glucose concentration in both treatments compared to controls. In an experimental infection with Vibrio anguillarum, fish previously exposed to subacute levels of ammonia were more susceptible to pathogenic challenge. The findings of this study indicate that a more thorough investigation into the effects of environmental ammonia on fish populations in coastal waters should be undertaken and the current environmental standards reassessed.

Population growth of Daphnia magna under multiple stress conditions: Joint effects of temperature, food, and cadmium

Aquatic organisms in the field often are exposed to combinations of stress factors of various origins. Little is known of the interaction between different types of stressors; hence, the predictability of their joint effects is low. Therefore, the present study analyzed the joint effects of temperature, food, and cadmium on the population growth rate of the water flea Daphnia magna. The results revealed that temperature, food, and cadmium, as well as their interactions, were important factors that influenced life-history parameters and, as a consequence, the population growth rate of D. magna. In general, population growth rate increased at high temperature and food level but decreased when cadmium was present. The positive effect of temperature on population growth rate was smallest at limiting food levels. Negative effects of cadmium on the growth rate were enhanced at elevated temperatures, whereas high food levels protected the daphnids from adverse effects of cadmium. To avoid over- or underestimation regarding the toxicity of substances to field populations, results of standard toxicity tests should be applied in a location-specific way.

Ecological impact in ditch mesocosms of simulated spray drift from a crop protection program for potatoes

Outdoor aquatic ditch mesocosms were treated with a range of pesticides to simulate various spray drift rates resulting from a typical crop protection program used in the cultivation of potatoes in The Netherlands. The main experimental aims of the present study were to provide information on the fate and ecological effects of drift of the pesticides into surface water and to evaluate the effectiveness of drift-reduction measures in mitigating risks. The pesticides selected and the dosage, frequency, and timing of application were based on normal agricultural practices in the potato crop. Applications of prosulfocarb, metribuzin (both herbicides), lambda-cyhalothrin (insecticide), chlorothalonil, and fluazinam (both fungicides) were made in the sequence typical of the spray calendar for potatoes. A total of 15 treatments with the various compounds were made by spray application to the water surface at 0.2%, 1%, and 5% of the recommended label rates. Chemical fate and effects on ecosystem function and structure (phytoplankton, zooplankton, chlorophyll-a, macroinvertebrates, macrophytes, breakdown of plant litter) were investigated. To interpret the observed effects, treatment concentrations were also expressed in toxic units (TU), which describe the relative toxicity of the compounds with standard toxicity test organisms (Daphnia and algae). After treatment, each compound disappeared from the water phase within 2 d, with the exception of prosulfocarb, for which 50% dissipation time (DT50) values ranged between 6 and 7 d. At the 5% treatment level, an exposure peak of 0.9 TUalgae was observed, which resulted in short-term responses of pH, oxygen, and phytoplankton. At the 5% treatment level, exposure concentrations also exceeded 0.1 TUDaphnia, and this resulted in long-term effects on zooplankton and macroinvertebrates, some of which did not fully recover by the end of the present study. At the 1% treatment level, only slight transient effects were observed on a limited number of zooplankton and macro-invertebrate species and on pH. At the 0.2% level, no consistent treatment-related effects were observed. Most of the observed effects were consistent with the results from higher-tier and mesocosm studies with the individual compounds. Multi and repeated stress played a small role within the applied pesticide package, because of rapid dissipation of most substances and the absence of many simultaneous applications. This suggests that risk assessments based on the individual compounds would in this case have been sufficiently protective for their uses in a crop protection program.

Toxicity benchmarks for antimony, barium, and beryllium determined using reproduction endpoints forFolsomia candida,Eisenia fetida, andEnchytraeus crypticus

The U.S. Environmental Protection Agency is developing ecological soil screening levels (Eco-SSLs) for the ecological risk assessment of contaminants at Superfund sites. The Eco-SSLs for several soil contaminants have been developed from toxicity benchmarks for soil invertebrates in the existing literature. Insufficient information to generate Eco-SSLs for Sb, Ba, and Be necessitated toxicity testing to fill the data gaps. We used standardized toxicity tests with the earthworm Eiseniafetida, enchytraeid Enchytraeus crypticus, and collembolan Folsomia candida in the present study. These tests were selected on the basis of their ability to measure chemical toxicity to ecologically relevant test species during chronic assays and their inclusion of at least one reproduction component among the measurement endpoints. Tests were conducted in Sassafras Sandy Loam soil, which supports relatively high bioavailability of metals. Weathering and aging procedures for metals in amended soil were incorporated into these studies to better reflect exposure conditions in the field. The relative toxicity of metals to the soil invertebrates tested was Be > Sb > Ba based on the median effective concentration values for reproduction. These studies produced toxicological data that can contribute to the development of Eco-SSLs for Sb, Ba, and Be for soil invertebrates.

Applying adult emergence as an endpoint in a post-exposure laboratory test using two midge species (Diptera: Chironomidae)

Several approaches have been used to evaluate biological impairment in aquatic ecosystems which can be categorized as either laboratory or field. In the recent years, the laboratory toxicity test approach has been extended to field exposures where ambient factors are allowed to influence the test response. Field exposures of laboratory test organisms require method modifications. In this paper, a novel in situ method is described which measures growth, survival and emergence of sediment inhabiting insects (Diptera: Chironomidae) that are used in standardized laboratory toxicity testing. Two standard chironomid species ( Chironomus riparius and Chironomus tentans) were used to test the suitability of the approach and to compare the performance of the species. The larvae were transferred to the laboratory for emergence after 7 days in situ exposure which was compared to laboratory responses. Growth, survival and emergence were significantly lower in the in situ pre-exposure than in the laboratory. Also, emergence success was significantly lower in one reference sediment (LMR) than in the other test sediments in both in situ and the laboratory treatment. These lower response levels likely resulted from sediment characteristics and artifacts related to the exposure in the in situ chamber. Feeding and water quality within the exposure chamber appear to be factors that may differ markedly from the laboratory exposure and may affect organism responses. C. riparius developed (growth, emergence time) faster than C. tentans in all treatments, otherwise the species responded similarly. C. riparius may be a better alternative for the chronic in situ exposures because of shorter exposure times and reduced feeding requirements.

Evaluation of Aquatic Toxicities of Chromium and Chromium-Containing Effluents in Reference to Chromium Electroplating Industries

This study evaluated aquatic toxicities of chromium and chromium-containing laboratory samples representative of effluents from chromium electroplating industries, and compared the aquatic environmental risks of hexavalent and trivalent chromium electroplating operations. Trivalent chromium electroplating has emerged as an acceptable alternative to hazardous hexavalent chromium electroplating. This process substitution has reduced the human health impact in the workplace and minimized the production of hazardous sludge regulated under the Resource Conservation and Recovery Act (RCRA). The thrust behind this research was to investigate whether trivalent chromium electroplating operations have lower adverse impacts on standardized toxicity test organisms. Ceriodaphnia dubia and Pimephales promelas were used to investigate toxicities of trivalent chromium (Cr (III)), hexavalent chromium (Cr (VI)), and industrial effluents. In agreement with previous studies, Cr (III) was found to be less toxic than Cr (VI). Despite having several organic and inorganic constituents in the effluents obtained from trivalent chromium plating baths, they exhibited less adverse effects to C. dubia than effluents obtained from hexavalent chromium electroplating baths. Thus, transition from hexavalent to trivalent chromium electroplating processes may be justified. However, because of the presence of organic constituents such as formate, oxalate, and triethylene glycol in effluents, trivalent chromium electroplating operations may face additional regulatory requirements for removal of total organic carbon.

An analysis of genetic toxicity, reproductive and developmental toxicity, and carcinogenicity data: I. Identification of carcinogens using surrogate endpoints

A retrospective analysis of standard genetic toxicity (genetox) tests, reproductive and developmental toxicity (reprotox) studies, and rodent carcinogenicity bioassays (rcbioassay) was performed to identify the genetox and reprotox endpoints whose results best correlate with rcbioassay observations. A database of 7205 chemicals with genetox ( n = 4961), reprotox ( n = 2173), and rcbioassay ( n = 1442) toxicity data was constructed; 1112 of the chemicals have both genetox and rcbioassay data and 721 chemicals have both reprotox and rcbioassay data. This study differed from previous studies by using conservative weight of evidence criteria to classify chemical carcinogens, data from 63 genetox and reprotox toxicological endpoints, and a new statistical parameter of correlation indicator (CI, the average of specificity and positive predictivity) to identify good surrogate endpoints for predicting carcinogenicity. Among 63 endpoints, results revealed that carcinogenicity was well correlated with certain tests for gene mutation ( n = 8), in vivo clastogenicity ( n = 2), unscheduled DNA synthesis assay ( n = 1), and reprotox ( n = 3). The current FDA regulatory battery of four genetox tests used to predict carcinogenicity includes two tests with good correlation (gene mutation in Salmonella and in vivo micronucleus) and two tests with poor correlation (mouse lymphoma gene mutation and in vitro chromosome aberrations) by our criteria.

Synthetic Musk Compounds and Effects on Human Health?

A recent article by Luckenbach and Epel (2005) on in vitro observations of inhibitory properties exhibited by certain nitromusk and polycyclic musk fragrance ingredients on mussel cells raised some concerns regarding potential environmental risks and safety to humans that may be associated with nitromusk and polycyclic musk compounds. The Research Institute for Fragrance Materials would like to address several points that may help readers more clearly understand the meaning and context of the reported research. The tonnages of musk compounds reported by Luckenbach and Epel (2005) in their article (7,000–8,000 tons) are higher than the industry-reported global tonnage of these materials. From 1995 to 2000, the total worldwide usage declined from 300 tons to 200 tons for musk xylene and musk ketone combined. The 2000 worldwide use of polycyclic musks is approximately 4,000 tons. Measured concentrations of these compounds in the environment are less than the effects concentrations reported by Luckenbach and Epel (2005). In a review of measured environmental concentrations, Rimkus (1999) stated that the highest reported measurement of hexahydrohexamethyl-cyclopenta (γ)-2-benzopyran (HHCB) in surface water was 12.5 μg/L (0.048 μM). The IC50 (concentration that inhibits 50%) reported for polycyclic musks was 2.34 μM. Overall, measured environmental concentrations were 2–6 orders of magnitude lower than the effects concentrations reported by Luckenbach and Epel (2005). The data reported by Luckenbach and Epel (2005) reflect a method under development. There are many steps between the observation of an in vitro effect and effects on whole organisms, ecosystems, and humans. In vivo studies in mussels and studies linking mussel gill tissue to undefined tissues in mammals and humans are some of the research necessary to conclude that these higher level effects may exist. These effects would then need to be placed into a risk-based context by comparing them to exposure concentrations. The safety of nitromusk and polycyclic musk compounds for humans has been extensively tested and affirmed by numerous regulatory agencies and academic scientists around the world [Scientific Committee on Cosmetic Products and Non-Food Products (SCCNFP) 2002a, 2002b]. The trace environmental levels of the musks continue to be investigated, and environmental safety and monitoring studies are ongoing so that the public can be assured of their safety. Regarding the environmental effects of synthetic musks, the IC10 (concentration that inhibits 10%) values should be compared to no observed effect concentrations (NOECs). The IC10 values of the synthetic musks are around the level of the lowest in vivo NOECs observed for aquatic organisms. Table 1 shows that the in vitro multi-xenobiotic resistance (MXR) transporter activity in mussel gill is of the same sensitivity as the effects observed in the standard toxicity tests with aquatic organisms. Thus, at the exposure level where the protective transporter efflux is decreased, rendering the cell more accessible to other potential toxicants, the effects of the synthetic musks are also indicated in other end points, such as development and growth. Table 1 Intensity of in vitro MXR transporter activity in mussel gill. The observed effects are not limited to the nitromusk and polycyclic musk compounds. For example, the other chemicals used by Luckenbach and Epel (2005)—verapamil and quinidine—also produced the phenomenon. In the case of verapamil, the IC10 was reported at 1–2 orders of magnitude below the nitromusks and polycyclic musks. I look forward to continued discussions with the Luckenbach and Epel to determine the relevance of the results of this study.

Behavioral response of Corophium volutator relative to experimental conditions, physical and chemical disturbances

The preference/avoidance behavioral response of a widely used amphipod in toxicity tests, Corophium volutator, was investigated in relation to the presence of anthropogenic physical or chemical materials in sediments. Exposure conditions, including the density of amphipods, the depth of sediments, amount of overlying water, and exposure time, were examined for their influence on amphipods' preference for field sediments and avoidance of coarse sand. It was shown that these variables did not affect the response; thus, conditions similar to published standard toxicity tests were chosen. A gradient of sediments spiked with potential habitat disturbances that can be found on a beach or in contaminated sediments, such as those in harbors, were tested. These substances included sand, seaweed, burned wood, coal, crankcase oil, and diesel oil. To enhance the interpretation of results and decrease the variability observed when tests were conducted at different times over the summer, exposures were performed over a gradient of spike material in reference sediments. We conclude that physical obstacles added to reference sediments lead to less correlation with the behavioral response than observed with chemical interferences. Amphipods' behavior ranked harbor sediments similarly to previous studies concerning the health of intertidal mussels collected in proximity to the sediments sites. For five sites, preference of reference sediments was observed until the level of polycyclic aromatic hydrocarbons in diluted harbor sediments reached the Canadian Council of Ministers of the Environment sediment quality guidelines.

Comparison of Plants for Germination Toxicity Tests in Petroleum-Contaminated Soils

Pollution of soil by petroleum hydrocarbons is a serious environmental problem world-wide. Although total concentration of contaminants in soil and/or water is used for regulatory review, it also is beneficial to assess the potential for ecosystem impact through a series of bioassays. One commonly used bioassay is seed germination. In this test, seeds are placed in contaminated material, and seedlings enumerated after a specified incubation period. However, different plant species produce variability in response. In the research project reported here, lettuce (Lactuca sativa L.), millet (Panicum miliaceum), radish (Raphanus L.), red clover (Trifolium pratense L.), and wheat (Triticum aestivum) were tested for sensitivity to petroleum-contaminated soil in germination tests. While most plants appeared to show some sensitivity to the pollutant, only lettuce had a statistically significant difference in response to contaminated and uncontaminated soil. These results confirm that Latuca sativa L is the optimal plant choice for standard germination toxicity tests with petroleum-impacted soil.

Development of a quantitative enzyme-linked immunosorbent assay for vitellin in the mysid Neomysis integer (Crustacea: Mysidacea)

Mysid crustaceans have been put forward by several regulatory bodies as suitable test organisms to screen and test the potential effects of environmental endocrine disruptors. Despite the well-established use of mysid reproductive endpoints such as fecundity, egg development time, and time to first brood release in standard toxicity testing, little information exists on the hormonal regulation of these processes. Control of vitellogenesis is being studied intensively because yolk is an excellent model for studying mechanisms of hormonal control, and vitellogenesis can be chemically disrupted. Yolk protein or vitellin is a major source of nourishment during embryonic development of ovigorous egg-laying invertebrates. The accumulation of vitellin during oocyte development is vital for the production of viable offspring. In this context, we developed a competitive enzyme-linked immunosorbent assay (ELISA) for vitellin of the estuarine mysid Neomysis integer. Mysid vitellin was isolated using gel filtration, and the purified vitellin was used to raise polyclonal antibodies. The ELISA was sensitive within a working range of 4 to 500 ng vitellin/mL. Serial dilutions of whole body homogenates from female N. integer and the vitellin standard showed parallel binding curves, validating the specificity of the ELISA. The intra- and interassay coefficients of variation were 8.2% and 13.8%, respectively. Mysid vitellin concentrations were determined from ovigorous females and eggs at different developmental stages. The availability of a quantitative mysid vitellin ELISA should stimulate further studies on the basic biology of this process in mysids. Furthermore, it could provide a means to better understand and predict chemically induced reproductive effects in mysids.

Life Stage Specific Impact of Dimethoate on the Predatory Mite Hypoaspis aculeifer Canestrini (Gamasida: Laelapidae)

Toxicants may affect juveniles more than adults because of physiological and behavioral aspects. When developing toxicity tests, this issue is often not addressed, and the most sensitive end point may be ignored. The topic was raised by a European working group aiming to develop a standard toxicity test with the predatory mite, Hypoaspis (Geolaelaps) aculeifer Canestrini, as this species was included in the EU Guidance documentfortesting of pesticides. To assess whether the juvenile life stages are the most susceptible, we examined the acute toxicity of dimethoate on larvae, protonymphs, deutonymphs, males, and females of H. aculeifer. The mites were exposed to 0, 2, 4, and 6 mg dimethoate kg(-1) for 7 days in an OECD artificial soil (5% organic matter). Total juvenile biomass, reproduction, mortality, and population growth rate (pgr) lambda were assessed at the end of the test. A comparison of mortality ranked the sensitivity of the life stages: Larvae (LC50 = 3.8 mg kg(-1)) > protonymphs (LC50 = 5.3 mg kg(-1)) > males (LC50 = 5.6 mg kg(-1)) > deutonymphs (LC50 = 7.1 mg kg(-1)) > females (LC50 = 7.6 mg kg(-1)). Effects on reproduction and pgr were significant at 2 mg dimethoate kg(-1), with population decline starting at this concentration. Thus, a test system with H. aculeifer including reproduction as end point is a rational approach, as reproduction will encompass juvenile mortality, at least with respect to dimethoate. Moreover, we suggest that pgr should be included in chronic standard tests because of high ecological relevance and the feasibility of applying it.

Effects of Dietary Administration of CL-20 on Japanese QuailCoturnix coturnix japonica

Hexanitrohexaazaisowurtzitane, or CL-20, is an emerging highly energetic compound currently under consideration for military applications. With the anticipated wide use of CL-20, there is the potential for soil and groundwater contamination resulting in adverse toxicologic effects on environmental receptors. Presently, there is a lack of data describing the toxic effects of CL-20 on avian species. The present study describes the effect of CL-20 on Japanese quail (Coturnix coturnix japonica) modified from standard toxicity test guidelines. First, a 14-day subacute assay was adopted using repeated gavage doses (0, 307, 964, 2439, 3475, or 5304 mg CL-20/kg body weight (BW)/d for 5 days followed by no CL-20 exposure (vehicle only) for 10 days. Second, a subchronic feeding assay (0, 11, 114, or 1085 mg CL-20/kg feed) was done for 42 days. During both studies, no overt toxicity was observed in the CL-20-treated birds. During the first 5 days of the subacute study, CL-20-exposed birds showed a dose-dependent decrease in BW gain, whereas increased liver weight, plasma sodium, and creatinine levels were observed in birds receiving the highest dose tested. For the subchronic study, embryo weights were significantly decreased in a dose-dependent manner. Embryos from CL-20-exposed birds were observed to have multiple cranial and facial deformities, beak curvatures, possible mid-brain enlargement, and classic one-sided development with micro-opthalamia (nonstatistical comparisons with control embryos). A trend toward decreased number of eggs laid per female bird was also observed. We conclude that CL-20 (or its degradation products) elicits few effects in adults but may affect avian development, although these preliminary findings should be confirmed.