Standard Toxicity Tests Research Articles

Effects of Soil Organic Matter Content on Cadmium Toxicity in Eisenia Fetida: Implications for the Use of Biomarkers and Standard Toxicity Tests

Bioavailability is affected by soil physicochemical characteristics such as pH and organic matter (OM) content. In addition, OM constitutes the energy source of Eisenia fetida, a well established model species for soil toxicity assessment. The present work aimed at assessing the effects of changes in OM content on the toxicity of Cd in E. fetida through the measurement of neutral red uptake (NRU) and mortality, growth, and reproduction (Organisation for Economic Co-operation and Development [OECD] Nos. 207 and 222). Complementarily, metallothionein (MT) and catalase transcription levels were measured. To decrease variability inherent to natural soils, artificial soils (Organization for Economic Cooperation and Development 1984) with different OM content (6, 10, and 14%) and spiked with Cd solutions at increasing concentrations were used. Low OM in soil decreased soil ingestion and Cd bioaccumulation but also increased Cd toxicity causing lower NRU of coelomocytes, 100 % mortality, and stronger reproduction impairment, probably due to the lack of energy to maintain protection mechanisms (production of MT).Cd bioaccumulation did not reflect toxicity, and OM played a pivotal role in Cd toxicity. Thus, OM content should be taken into account when using E. fetida in in vivo exposures for soil health assessment.

Safety assessment of the butyrate-producing Butyricicoccus pullicaecorum strain 25-3T, a potential probiotic for patients with inflammatory bowel disease, based on oral toxicity tests and whole genome sequencing

Inflammatory bowel disease (IBD) is a chronic inflammation of the digestive tract, characterized by dysbiosis of the intestinal microbiota. Probiotics have been suggested as a strategy to reduce active disease or extend remission. We isolated and characterized the butyrate-producing strain Butyricicoccus pullicaecorum 25-3T and identified it as a potential probiotic for patients with IBD. To evaluate the safety of 25-3T for use in humans, we conducted a standard acute oral toxicity test and a 28-day repeated oral dose toxicity test. The complete genome of B. pullicaecorum 25-3T was sequenced to search for virulence factors and antibiotic resistance determinants. The minimum inhibitory concentration (MIC) of 21 antimicrobials was determined. Results showed no adverse effects in the oral toxicity tests. B. pullicaecorum 25-3T is resistant against aminoglycosides and trimethoprim. The genome of 25-3T contains no virulence factors, one gene related to harmful metabolites and 52 sequences with high similarity to antimicrobial and toxic compound resistance genes, that did not correspond with a resistant phenotype. This first report of a safety assessment of a butyrate-producing strain from Clostridium cluster IV shows that B. pullicaecorum 25-3T is a non-pathogenic strain, but carries antibiotic resistance genes with the risk of transfer, that need further investigation.

Differences in soil solution chemistry between soils amended with nanosized CuO or Cu reference materials: implications for nanotoxicity tests.

Soil toxicity tests for metal oxide nanoparticles often include micrometer-sized oxide and metal salt treatments to distinguish between toxicity from nanometer-sized particles, non-nanometer-sized particles, and dissolved ions. Test result will be confounded if each chemical form has different effects on soil solution chemistry. We report on changes in soil solution chemistry over 56 days-the duration of some standard soil toxicity tests-in three soils amended with 500 mg/kg Cu as nanometer-sized CuO (nano), micrometer-sized CuO (micrometer), or Cu(NO3)2 (salt). In the CuO-amended soils, the log Cu2+ activity was initially low (minimum -9.48) and increased with time (maximum -5.20), whereas in the salt-amended soils it was initially high (maximum -4.80) and decreased with time (minimum -6.10). The Cu2+ activity in the nano-amended soils was higher than in the micrometer-amended soils for at least the first 11 days, and lower than in the salt-amended soils for at least 28 d. The pH, and dissolved Ca and Mg concentrations in the CuO-amended soils were similar, but the salt-amended soils had lower pH for at least 14 d, and higher Ca and Mg concentrations throughout the test. Soil pretreatments such as leaching and aging prior to toxicity tests are suggested.

Response of the green alga Oophila sp., a salamander endosymbiont, to a PSII‐inhibitor under laboratory conditions

In a rare example of autotroph-vertebrate endosymbiosis, eggs of the yellow-spotted salamander (Ambystoma maculatum) are colonized by a green alga (Oophila sp.) that significantly enhances salamander development. Previous studies have demonstrated the potential for impacts to the salamander embryo when growth of the algae is impaired by exposure to herbicides. To further investigate this relationship, the authors characterized the response of the symbiotic algae (Oophila sp.) alone to the photosystem II (PSII) inhibitor atrazine under controlled laboratory conditions. After extraction of the alga from A. maculatum eggs and optimization of culturing conditions, 4 toxicity assays (96 h each) were conducted. Recovery of the algal population was also assessed after a further 96 h in untreated media. Average median effective concentration (EC50) values of 123 µg L(-1) (PSII yield), 169 µg L(-1) (optical density), and 299 µg L(-1) (growth rate) were obtained after the 96-h exposure. Full recovery of exposed algal populations after 96 h in untreated media was observed for all endpoints, except for optical density at the greatest concentration tested (300 µg L(-1) ). Our results show that, under laboratory conditions, Oophila sp. is generally less sensitive to atrazine than standard test species. Although conditions of growth in standard toxicity tests are not identical to those in the natural environment, these results provide an understanding of the tolerance of this alga to PSII inhibitors as compared with other species.

Toxic potential of paracetamol to freshwater organisms: A headache to environmental regulators?

Paracetamol is one of the most prescribed drugs globally, due to its antipyretic and analgesic properties. However, it is highly toxic at elevated doses, with involvement of an already described oxidative stress pathway. Despite this, the number of ecotoxicological studies on potential effects of paracetamol in wild organisms is still scarce. The present article presents a comprehensive series of standardized assays for the assessment of paracetamol effects in freshwater organisms. The results show that paracetamol toxicity is widely variable among species, even when these species are phylogenetically related. Furthermore, comparisons between data from the literature and our results reinforce this conclusion, providing evidence of the inadequacy of standardized toxicity testing guidelines for pharmaceutical compounds in wild organisms. Paracetamol toxicity can be modulated by unpredictable physiological conditions that might compromise extrapolations and comparisons of responsiveness among species. The ecological relevance of data obtained from classical tests for this compound is further discussed.

Toxicity and repellency of dimethoate, pirimiphos-methyl and deltamethrin against Tribolium castaneum (Herbst) using different exposure methods

Tribolium castaneum, the red flour beetle, is one of the major pests of cereal grains and their products. Infestation of raw food products by T. castaneum can be tackled by use of insecticides which offer a rapid and effective control method for different insect pests. In the present study, toxicity and repellency of three widely used insecticides was assessed on adult red flour beetles to investigate how a flour substrate affects the results of the standard laboratory toxicity and repellency test methods. The red flour beetles were exposed to the organophosphates dimethoate and pirimiphos-methyl and the pyrethroid deltamethrin, following the original and two modified residual film methods (toxicity assessment) and by the area preference method to assess repellency. Since the conditions of exposure (glass and filter paper surfaces as exposure substrates) in the original methods are quite different compared to the exposure carried out in stored-product protection, methods were modified to include flour as an exposure substrate. The results showed that according to the LC50 values, toxicity of the investigated insecticides could be arranged in the following order: pirimiphos-methyl > dimethoate > deltamethrin. Also, both mortality and repellency were dependent on the exposure methods, i.e. presence and treatment of flour substrate. Mortality was significantly reduced in comparison to the original method. Repellency was recorded when beetles were exposed to deltamethrin using flour as a substrate, whereas using the filter paper surface (original method) repellency was not obtained. The results of the present study clearly indicate that there is an influence of substrate on the susceptibility of T. castaneum to insecticides and that the efficacy of different insecticides is affected by exposure substrate.

Application of microcosmic system for assessment of insecticide effects on biomarker responses in ecologically different earthworm species

Earthworms from different ecological categories—epigeic Eisenia andrei and Lumbricus rubellus, endogeic Octolasion lacteum and anecic Lumbricus terrestris—were exposed in a microcosmic system to three commonly used insecticides. The effects of the insecticides were evaluated by measuring the following molecular biomarkers—the activities of AChE, CES, CAT, GST and the concentration of GSH. The results showed that environmentally relevant doses of organophosphates dimethoate and pirimiphos-methyl significantly affected the measured biomarkers, whereas pyrethroid deltamethrin did not affect the earthworms at the recommended agricultural dose. Considering the ecological category of earthworms, the results were inhomogeneous and species-specific differences in the biomarker responses were recorded. Since the biomarker responses of the investigated earthworm species were different after exposure to organophosphates in a microcosm compared to the exposure via standardized toxicity tests, two types of species sensitivity should be distinguished—physiological and environmental sensitivity. In addition, the hormetic effect of organophosphates on AChE and CES activities was recorded. The detection of hormesis in a microcosm is of great importance for future environmental research and soil biomonitoring, since in a realistic environment pollutants usually occur at low concentrations that could cause a hormetic effect. The results demonstrate the importance of the application of microcosmic systems in the assessment of the effects of environmental pollutants and the necessity of taking into account the possible differences between physiological and environmental species sensitivity.

Issues and challenges with oil toxicity data and implications for their use in decision making: A quantitative review

Aquatic toxicity considerations are part of the net environmental benefit analysis and approval decision process on the use of dispersants in the event of an offshore oil spill. Substantial information is available on the acute toxicity of physically and chemically dispersed oil to a diverse subset of aquatic species generated under controlled laboratory conditions. However, most information has been generated following standard laboratory practices, which do not realistically represent oil spill conditions in the field. The goal of the present quantitative review is to evaluate the use of standard toxicity testing data to help inform decisions regarding dispersant use, recognizing some key issues with current practices, specifically, reporting toxicity metrics (nominal vs measured), exposure duration (standard durations vs short-term exposures), and exposure concentrations (constant vs spiked). Analytical chemistry data also were used to demonstrate the role of oil loading on acute toxicity and the influence of dispersants on chemical partitioning. The analyses presented here strongly suggest that decisions should be made, at a minimum, based on measured aqueous exposure concentrations and, ideally, using data from short-term exposure durations under spiked exposure concentrations. Available data sets are used to demonstrate how species sensitivity distribution curves can provide useful insights to the decision-making process on dispersant use. Finally, recommendations are provided, including the adoption of oil spill-appropriate toxicity testing practices.

Facilitating the use of non-standardin vivostudies in health risk assessment of chemicals: a proposal to improve evaluation criteria and reporting

To improve data availability in health risk assessment of chemicals and fill information gaps there is a need to facilitate the use of non-standard toxicity studies, i.e. studies not conducted according to any standardized toxicity test guidelines. The purpose of this work was to propose criteria and guidance for the evaluation of reliability and relevance of non-standard in vivo studies, which could be used to facilitate systematic and transparent evaluation of such studies for health risk assessment. Another aim was to propose user friendly guidance for reporting of non-standard studies intended to promote an improvement in reporting of studies that could be of use in risk assessment. Requirements and recommendations for the design and execution of in vivo toxicity studies were identified from The Organisation for Economic Co-operation and Development (OECD) test guidelines, and served as basis for the data evaluation criteria and reporting guidelines. Feedback was also collected from experts within the field of toxicity testing and risk assessment and used to construct a two-tiered framework for study evaluation, as well as refine the reporting guidelines. The proposed framework emphasizes the importance of study relevance and an important aspect is to not completely dismiss studies from health risk assessment based on very strict criteria for reliability. The suggested reporting guidelines provide researchers with a tool to fulfill reporting requirements as stated by regulatory agencies. Together, these resources provide an approach to include all relevant data that may fill information gaps and reduce scientific uncertainty in health risk assessment conclusions, and subsequently also in chemical policy decisions.

Exposures to a selective serotonin reuptake inhibitor (SSRI), sertraline hydrochloride, over multiple generations: Changes in life history traits in Ceriodaphnia dubia

Selective serotonin reuptake inhibitors (SSRIs) have been reported to range from low parts per trillion to parts per billion levels in surface waters, wastewater effluents, and sediments. These low levels have led to concern for their potential long-term risks to the survival, growth, and reproduction of aquatic organisms. We investigated the acute and chronic effects of sertraline on the life history traits of Ceriodaphnia dubia over the course of three generations under environmentally realistic concentrations. Acute toxicity of sertraline in C. dubia offspring resulted in a 48h median effective concentration of 126µgL−1. Under chronic exposure, the lowest concentration to affect fecundity and growth was at 53.4µgL−1 in the first two generations. These parameters become more sensitive during the third generation where the LOEC was 4.8µgL−1. The median effective concentrations (EC50) for the number of offspring per female, offspring body size, and dry weight were 17.2, 21.2, and 26.2µgL−1, respectively. Endpoints measured in this study demonstrate that chronic exposure of C. dubia to sertraline leads to effects that occur at concentrations only an order of magnitude higher than predicted environmental concentrations. However, this study also demonstrates that multigenerational effects should be considered in chronic exposure studies because standard toxicity tests do not account for increases in sensitivity in successive generations to toxicants.

An emerging energetic soil contaminant, CL-20, can affect the soil invertebrate community in a sandy loam soil

We investigated the effects of nitramine explosive CL-20 (China Lake compound 20) on the indigenous soil invertebrate community in Sassafras sandy loam (SSL) soil using a 12-week soil microcosm assay. Freshly collected SSL soil was amended with CL-20 to prepare multiple treatment concentrations ranging from 0 (acetone control) to 10,300mgkg−1. The selected concentration range of CL-20 adequately assessed the concentration–response relationships for total microarthropods, and for individual microarthropod groups. The overall composition of microarthropod community in SSL soil was not affected by exposure to CL-20, based on the number of taxonomic groups present in the individual treatments after 12 weeks. However, community structure analysis revealed greater sensitivity to CL-20 by predatory mesostigmatid mites. Microarthropod and nematode communities showed contrasting sensitivities to CL-20 in SSL soil. Total numbers of nematodes were either unaffected or significantly (p<0.05) increased in CL-20 treatments compared with control. Only predator group among nematodes was consistently adversely affected by exposure to CL-20. The abundance of predatory nematodes decreased in a concentration-dependent manner throughout the 12-week exposure. Microcosm assay with corresponding community structure analysis can provide the means for validating the ecotoxicity data from standardized laboratory tests, both complimenting and expanding upon the ecotoxicological significance of data from standardized single-species toxicity tests.

Determining the minimum effective dose of rotenone for eradication of alien smallmouth bass Micropterus dolomieu from a South African river

In February 2012 the Rondegat River, in the Cape Floristic Region, was the first river in South Africa where the piscicide rotenone was used to remove an alien invasive fish, smallmouth bass Micropterus dolomieu. In preparation for this treatment, the sensitivity of smallmouth bass to various concentrations of the rotenone formulation CFT Legumine (5% active rotenone) was evaluated a week prior to treatment using standard toxicity tests to determine the minimum effective dose (MED) that would result in 100% mortality after exposure for 4 h. The MED was 0.0125 mg l-1 rotenone. Adverse effects, including erratic swimming, loss of equilibrium and death, occurred in a dose-dependent manner with smaller fish responding faster than larger ones. Standard operating procedures for the use of rotenone recommend treatment at a minimum of twice the calculated MED. Given the uncertainty associated with rotenone losses through hydrolysis and photolysis under field conditions, and the possible occurrence of smallmouth bass more tolerant than those tested, a concentration of twice the recommended treatment dose (0.050 mg l-1 rotenone) was finally used to treat the Rondegat River for a duration of 6 h.

Assessment of toxicity test endpoints for freshwater mussel larvae (glochidia)

The objectives of the present study were to determine if the viability of freshwater mussel larvae (glochidia) is an ecologically relevant endpoint for toxicity tests and to define the appropriate duration of those tests. The authors assessed 1) how viability (the shell closure response to sodium chloride) compares with infectivity (ability to attach to a host fish and successfully metamorphose to the juvenile stage), and 2) the decline of viability and infectivity over time after glochidia were released from female mussels. Glochidia of 7 mussel species were isolated from females, placed in water, and subsampled daily for 2 d to 5 d. Viability, when ≥90%, was generally a good predictor of infectivity; however, when viability was <90%, infectivity was often disproportionately low, especially for glochidia collected near the end of the brooding period. Viability and infectivity declined more rapidly in natural water and sediment compared to reconstituted water. Following 24-h exposure to a toxicant (sodium chloride or copper), infectivity of the viable glochidia did not differ among concentrations of toxicants. The results indicate that viability is a valid proxy for infectivity and an ecologically relevant endpoint for standard toxicity tests with freshwater mussels for any test duration with control viability >90%.

Analysis of the ecotoxicity data submitted within the framework of the REACH Regulation: Part 4. Experimental terrestrial toxicity assays

This paper summarises the terrestrial ecotoxicity data submitted in the REACH11Registration, Evaluation, Authorisation and Restriction of Chemicals. registration dossiers and disseminated by ECHA.22European Chemicals Agency. The analysis describes both the guidelines and the test species mostly used by registrants. REACH information requirements in relation to the effects on terrestrial organisms encompass three trophic levels; invertebrates, plants and micro-organisms, and the study of both long and short-term exposure. The results observed for soil invertebrates showed that on one hand there was a clear prevalence for testing on the species recommended by the standard test guidelines. On the other, the reporting included a large variety of species from very different families, demonstrating the feasibility for conducting toxicity tests on a number of relevant groups e.g. for species sensitivity distribution approaches. Standard toxicity testing with terrestrial plants under REACH follows a different approach and requires simultaneous testing on several species, using the same test conditions, adapted to each species, if needed. The test methods used to conduct the studies were only reported for 30% of cases. The most extensively reported test guidelines for terrestrial plants were OECD33Organisation for Economic Cooperation and Development. 208, ISO44International Organisation for Standardisation. 11269-1 and ISO 11269-1. Information requirements for soil micro-organisms under REACH are related to the analysis of functional endpoints instead of on species or taxa. As recommended in REACH, OECD 216 and OECD 217 were the most often used test methods for soil micro-organisms. But overall, the test method was reported for only about 40% of the experimental studies. Moreover, it is noted that information on potential effects on soil micro-organisms is available for a limited number of REACH registered substances. The assessment suggests that providing waiving justifications and collecting available information, which in many cases might be well used for covering standard REACH data requirements, have been the main approaches used by registrants for the first REACH registration deadline.

Insulin Resistance and Environmental Pollutants: Experimental Evidence and Future Perspectives

Background: The metabolic disruptor hypothesis postulates that environmental pollutants may be risk factors for metabolic diseases. Because insulin resistance is involved in most metabolic diseases and current health care prevention programs predominantly target insulin resistance or risk factors thereof, a critical analysis of the role of pollutants in insulin resistance might be important for future management of metabolic diseases.Objectives: We aimed to critically review the available information linking pollutant exposure to insulin resistance and to open the discussion on future perspectives for metabolic disruptor identification and prioritization strategies.Methods: We searched PubMed and Web of Science for experimental studies reporting on linkages between environmental pollutants and insulin resistance and identified a total of 23 studies as the prime literature.Discussion: Recent studies specifically designed to investigate the effect of pollutants on insulin sensitivity show a potential causation of insulin resistance. Based on these studies, a summary of viable test systems and end points can be composed, allowing insight into what is missing and what is needed to create a standardized insulin resistance toxicity testing strategy.Conclusions: It is clear that current research predominantly relies on top-down identification of insulin resistance–inducing metabolic disruptors and that the development of dedicated in vitro or ex vivo screens to allow animal sparing and time- and cost-effective bottom-up screening is a major future research need.Citation: Hectors TL, Vanparys C, Van Gaal LF, Jorens PG, Covaci A, Blust R. 2013. Insulin resistance and environmental pollutants: experimental evidence and future perspectives. Environ Health Perspect 121:1273–1281; http://dx.doi.org/10.1289/ehp.1307082

Assessing sediment toxicity: Past, present, and future

Assessing sediment toxicity: Past, present, and future

An ecotoxicological analysis of the sediment quality in a European Atlantic harbor emphasizes the current limitations of the Water Framework Directive

The “PortoNovo” project was developed to standardize the methodologies for water quality management in the port areas of coastal Atlantic regions to improve the Water Frame Directive (WFD) for these specific water bodies. Under this scope, water and sediment samples were collected from five sites within the Port of Aveiro, Portugal. According to the physical and chemical parameters that were analyzed (i.e., metals, total organic carbon, polychlorinated biphenyls and polycyclic aromatic hydrocarbons), the sediments were not considered at risk based on European sediment quality laws. However, the bioassays that were performed on the sediment samples (Microtox®) and the standardized acute toxicity test using the marine rotifer, Brachionus plicatilis, on sediment elutriates revealed higher toxicity levels. The use of bioassays to assess sediment quality clearly complements more conservative approaches and highlights current gaps within the WFD. The approach presented here can be easily transferred to other port areas for more reliable water quality management.

Comparison of effects on crustaceans: carbon nanoparticles and molybdenum compounds nanowires

Carbon nanomaterials (CNM) and molybdenum compound nanostructures are materials with various applications yet little is known regarding the toxicity of these nanoparticles in pristine form in aquatic environment. Daphnia magna standard acute toxicity test (EN ISO 6341:1996; freshwater) and Artemia salina standard acute toxicity test (ArtoxKit standard method; 15 ppt saltwater) were applied to assess the toxicity of non-modified CNM and molybdenum compound nanowires in water. It has been observed that CNM are more toxic in freshwater suspensions and somewhat more toxic than the tested molybdenum compound nanowires.

Evaluation of Daphniamagna for the Ecotoxicity Assessment of Alkali Leachate from Concrete

The cladoceran Daphniamagna has been used as an aquatic test species in aquatic toxicology. To evaluate the aquatic toxicity of leachate from concrete, the immobilization of D. magna was observed after treatment of various concentrations of leachate specimens. Reliabilities of the culture condition and the experimental protocol for acute toxicity test were successfully achieved from the standard toxicity test. The leachates were prepared from the mixture of Ordinary Portland Cement (OPC) and pozzolanic admixtures, Pulverised fuel ash (PFA), Ground granulated blast furnace slag (GGBS) and GGBS containing loess. Acute toxicity test showed 100% immobilization of D. magna for OPC or PFA. The leachates from OPC or PFA had high pH 10 to 12. However, GGBS and GGBS containing loess showed less toxicity according to the concentrations. Especially, immobilization was not observed at the concentrations below 12.5% of GGBS containing loess. Also the range of pH for these specimens was 8 to 9. This suggested that the use of loess as the admixture in concrete may be useful to reduce eco-toxicity of leachates from concrete. This our study provided the harmfulness of the alkali leaching from concrete in aquatic environment and the usefulness of D. magna to evaluate the toxicity of leachates from concrete.

Life-stage-dependent sensitivity of the cyclopoid copepod Mesocyclops leuckarti to triphenyltin

The effects of triphenyltin (TPT) on non-target aquatic groups like copepods have not yet been studied in detail. Recently, Mesocyclops leuckarti was suggested as a representative species for freshwater cyclopoid copepods to study the effects of plant protection products. We conducted 96h acute toxicity tests in the laboratory to study the effects of TPT on the different life stages of M. leuckarti. We used the recently proposed General Unified Threshold model of Survival (GUTS) to mechanistically describe the underlying toxicokinetic and toxicodynamic process for the nauplii, copepodites and adults (males and females) of M. leuckarti. Experimental results showed that nauplii were significantly more sensitive to TPT compared to the other stages. Also, the 96h LC50 values were much lower than the 48h LC50 values, suggesting that standard acute toxicity test durations for aquatic invertebrates may underestimate toxic effects with respect to compounds like TPT which have a slow mechanism of action. The GUTS analysis, which mechanistically described the toxicity of TPT to the different life stages of M. leuckarti, suggested that the differences in sensitivity were a consequence of a lower threshold of effects in the nauplii compared to a higher threshold in the later stages. This study shows the potential of using the GUTS to understand how toxic compounds interact with different life-stages. Further experiments to provide a more detailed understanding of toxicokinetics of TPT in copepods could be useful.