Standard Acute Toxicity Tests Research Articles

Comparative toxicity assessment of alternative versus legacy PFAS: Implications for two primary trophic levels in freshwater ecosystems

Perfluoroalkyl substances (PFAS) are common environmental pollutants, but their toxicity framework remains elusive. This research focused on ten PFAS, evaluating their impacts on two ecotoxicologically relevant model organisms from distinct trophic levels: the crustacean Daphnia magna and the unicellular green alga Raphidocelis subcapitata. The results showed a greater sensitivity of R. subcapitata compared to D. magna. However, a 10-day follow-up to the 48 h immobilisation test in D. magna showed delayed mortality, underlining the limitations of relying on EC50 s from standard acute toxicity tests. Among the compounds scrutinized, Perfluorodecanoic acid (PFDA) was the most toxic to R. subcapitata, succeeded by Perfluorooctane sulfonate (PFOS), Perfluorobutanoic acid (PFBA), and Perfluorononanoic acid (PFNA), with the latter being the only one to show an algicidal effect. In the same species, assessment of binary mixtures of the compounds that demonstrated high toxicity in the single evaluation revealed either additive or antagonistic interactions. Remarkably, with an EC50 of 31 mg L-1, the short-chain compound PFBA, tested individually, exhibited toxicity levels akin to the notorious long-chain PFOS, and its harm to freshwater ecosystems cannot be ruled out. Despite mounting toxicological evidence and escalating environmental concentrations, PFBA has received little scientific attention and regulatory stewardship. It is strongly advisable that regulators re-evaluate its use to mitigate potential risks to the environmental and human health.

Environmental RNA as a Noninvasive Tool for Assessing Toxic Effects in Fish: A Proof-of-concept Study Using Japanese Medaka Exposed to Pyrene.

Although environmental RNA (eRNA) is emerging as a noninvasive tool to assess the health status of aquatic macroorganisms, the potential of eRNA in assessing chemical hazards remain largely untested. In this study, we investigated the ability of eRNA to detect changes in gene expression in Japanese medaka fish (Oryzias latipes) in response to sublethal pyrene exposure, as a model toxic chemical. We performed standardized acute toxicity tests and collected eRNA from tank water and RNA from fish tissue after 96 h of exposure. Our results showed that over 1000 genes were detected in eRNA and the sequenced read counts of these genes correlated with those in fish tissue (r = 0.50). Moreover, eRNA detected 86 differentially expressed genes in response to pyrene, some of which were shared by fish RNA, including the suppression of collagen fiber genes. These results suggest that eRNA has the potential to detect changes in gene expression in fish in response to environmental stressors without the need for sacrificing or causing pain to fish. However, we also found that the majority of sequenced reads of eRNA (>99%) were not mapped to the reference medaka genome and they originated from bacteria and fungi, resulting in low sequencing depth. In addition, eRNA, in particular nuclear genes, was highly degraded with a median transcript integrity number (TIN) of <20. These limitations highlight the need for future studies to improve the analytical methods of eRNA application.

Age and Synchronization of Daphnia magna Affect Sensitivity to Teflubenzuron in Acute Standardized Toxicity Tests.

The standard Daphnia sp. acute toxicity test for assessing the adverse effects of chemicals on aquatic invertebrates stipulates the use of neonates that are ≤24 h old (hours post release [hpr]) at the start of the exposure. However, when one is assessing acute effects of chemicals interfering with endocrine relevant-processes such as molting, both age synchronization and absolute age can influence the test outcome, because the occurrence of molting and associated mortality is highly time specific. Hence, a 24-h age synchronization window may mask the real effects of these compounds. To explore the influence of age synchronization and absolute age in standard acute toxicity tests, we exposed D. magna from different synchronization windows and absolute ages (≤4, 4-8, 8-12, ≤12, and ≤24 hpr at the beginning of the exposure) to 0.5-12 µg/L of the chitin synthesis inhibitor (CSI) teflubenzuron (TEF) using the Organisation for Economic Co-operation and Development test guideline 202 (Daphnia sp. 48 h immobilization test). Our results show significant differences in 48-h median lethal concentrations between animals with a synchronization window of ≤4 hpr (2.9 µg/L) and longer synchronization windows such as ≤12 hpr (5.1 µg/L) and ≤24 hpr (16.8 µg/L). A concurrent decreasing trend in molting median effect concentrations was observed for the same synchronization windows: ≤4 hpr (4.0 µg/L), ≤12 hpr (5.9 µg/L), and ≤24 hpr (30.0 µg/L). Together, our results show that both synchronization and absolute age are determinant factors for the sensitivity of D. magna to TEF. A narrow synchronization window (e.g., ≤4 hpr) may provide a more conservative estimate of TEF toxicity and should be considered when one is performing standardized toxicity tests for molting-disrupting compounds such as TEF. Environ Toxicol Chem 2023;42:1806-1815. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Evaluation of in vitro toxicity information for zebrafish as a promising alternative for chemical hazard and risk assessment

In vitro assays are widely proposed as a test alternative to traditional in vivo standard acute and chronic toxicity tests. However, whether toxicity information derived from in vitro assays instead of in vivo tests could provide sufficient protection (e.g., 95 % of protection) for chemical risks remain evaluated. To investigate the feasibility of zebrafish (Danio rerio) cell-based in vitro test method as a test alternative, we comprehensively compared sensitivity differences among endpoints, among test methods (in vitro, FET and in vivo), and between zebrafish and rat (Rattus norvegicus), respectively using chemical toxicity distribution (CTD) approach. For each test method involved, sublethal endpoints were more sensitive than lethal endpoints for both zebrafish and rat, respectively. Biochemistry (zebrafish in vitro), development (zebrafish in vivo and FET), physiology (rat in vitro) and development (rat in vivo) were the most sensitive endpoints for each test method. Nonetheless, zebrafish FET test was the least sensitive one compared to its in vivo and in vitro tests for either lethal or sublethal responses. Comparatively, rat in vitro tests considering cell viability and physiology endpoints were more sensitive than rat in vivo test. Zebrafish was found to be more sensitive than rat regardless of in vivo or in vitro tests for each pairwise endpoint of concern. Those findings indicate that zebrafish in vitro test is a feasible test alternative to zebrafish in vivo and FET test and traditional mammalian test. It is suggesting that zebrafish in vitro test can be optimized by choosing more sensitive endpoints, such as biochemistry to provide sufficient protection for zebrafish in vivo test and to establish applications of zebrafish in vitro test in future risk assessment. Our findings are vital for evaluating and further application of in vitro toxicity toxicity information as an alternative for chemical hazard and risk assessment.

Size- and sex-related sensitivity differences of aquatic crustaceans to imidacloprid

Field collected aquatic invertebrates are often used as test organisms in the refinement of the standard Tier 1 risk assessment of various pollutants. This approach can provide insights into the effects of pollutants on the natural environment. However, researchers often pragmatically select test organisms of a specific sex and/or size, which may not represent the sensitivity of the whole population. To investigate such intraspecies sensitivity differences, we performed standard acute toxicity and toxicokinetic tests with different size classes and sex of Gammarus pulex and Asellus aquaticus. Furthermore, toxicokinetics and toxicodynamics models were used to understand the mechanism of the intraspecies sensitivity differences. We used neonates, juveniles and male and female adults in separate dedicated experiments, in which we exposed the animals to imidacloprid and its bioactive metabolite, imidacloprid-olefin. For both species, we found that neonates were the most sensitive group. For G. pulex, the sensitivity decreased linearly with size, which can be explained by the size-related uptake rate constant in the toxicokinetic process and size-related threshold value in the toxicodynamic process. For A. aquaticus, female adults were least sensitive to imidacloprid, which could be explained by a low internal biotransformation of imidacloprid to imidacloprid-olefin. Besides, imidacloprid-olefin was more toxic than imidacloprid to A. aquaticus, with differences being 8.4 times for females and 2.7 times for males. In conclusion, we established size-related sensitivity differences for G. pulex and sex-related sensitivity for A. aquaticus, and intraspecies differences can be explained by both toxicokinetic and toxicodynamic processes. Our findings suggest that to protect populations in the field, we should consider the size and sex of focal organisms and that a pragmatic selection of test organisms of equal size and/or sex can underestimate the sensitivities of populations in the field.

Towards the Development of Standardized Bioassays for Corals: Acute Toxicity of the UV Filter Benzophenone-3 to Scleractinian Coral Larvae.

Coral reefs have been declining globally at a historically unprecedented rate. Ultraviolet (UV) filters used in sunscreens may contribute to this decline at local scales, which has already led to bans on various organic UV filters in some regions. However, the underlying studies for these bans demonstrated significant flaws in the experimental design due to a lack of validated and standardized testing methods for corals. This study aimed to investigate options for the development of a standard acute toxicity test for the larval stage of scleractinian corals. Planula larvae of two brooding (Leptastrea purpurea and Tubastraea faulkneri) and two spawning (Acropora digitifera and A. millepora) species were exposed to the organic UV filter benzophenone-3 (BP3) for 48 h under static conditions. We observed interspecific variations in toxicity, with A. digitifera being the most sensitive (LC50 = 0.75 µg L−1) and T. faulkneri the least sensitive (LC50 = 2951.24 µg L−1) species. Inhibition of settlement was found to be a useful endpoint leading to an EC50 of 1.84 µg L−1 in L. purpurea larvae. Although the analytical challenges of measuring lipophilic substances in small volume test setups remain, the here applied test design and selected endpoints are suitable for further validation and subsequent standardization.

Assessment of surface water quality of the bois river (Goiás, Brazil) using an integrated physicochemical, microbiological and ecotoxicological approach

The data on water pollution is scarce in developing countries, including Brazil. The water quality assessment is important implementing the monitoring and remediation programs to minimize the risk of hazardous substances in freshwaters. Thus, this study evaluated the surface water quality of a stretch of the Bois River (Brazil), based on the physicochemical, microbiological and ecotoxicological analyses conducted in 2017, using Standard Methods and fish embryo acute toxicity (FET) test with zebrafish (Danio rerio). The results indicated that the quality of water samples located close to the discharge of tannery effluents was most impaired. Total phosphorus, BOD, DO, ammoniacal nitrogen, and thermotolerant coliforms parameters in P4 were not in accordance with the standards of current Brazilian legislation. Iron, lead, and copper levels were higher than environmental standards. The physicochemical quality of water samples was lower in the dry season than the rainy season. All samples (P1, P3, and P5) in rainy and dry seasons did not induce significant acute toxicity for zebrafish early-life stage; however other trophic levels (algae and microcrustacean) should be investigated to gain a better understanding of the toxicity during water quality analysis. In conclusion, the physicochemical and microbiological changes in the water of the Bois River can affect aquatic organisms as well as humans when it is used for drinking or in agriculture.

Daphnia magna and Ceriodaphnia dubia Have Similar Sensitivity in Standard Acute and Chronic Toxicity Tests.

The cladocerans Daphnia magna and Ceriodaphnia dubia have been used for decades to assess the hazards of chemicals and effluents, but toxicity data for these species have traditionally been treated separately. Numerous standard acute and chronic test guidelines have been developed for both species. In the present study, data were compiled and curated for acute survival (48 h) and growth and reproduction tests with D. magna (21 days chronic) and C. dubia (7 days chronic) toxicity assays. Orthogonal regressions were developed to statistically compare the acute and chronic sensitivity of D. magna and C. dubia across a diversity of chemicals and modes of action. Acute orthogonal regressions between D. magna and D. pulex, a widely accepted surrogate species, were used to set a data-driven benchmark for what would constitute a suitable D. magna surrogate. The results indicate that there is insufficient evidence to suggest a difference in acute or chronic sensitivity of D. magna and C. dubia in standard toxicity tests. Further, the variability in the acute D. magna and C. dubia regressions were of the same magnitude as that in D. magna and D. pulex regressions. Slope and y-intercept values were also comparable. The absence of significant differences in toxicity values suggests similar species sensitivity in standard tests across a range of chemical classes and modes of action. Environ Toxicol Chem 2022;41:134-147. © 2021 SETAC.

Evaluation of Juvenile Freshwater Mussel Sensitivity to Multiple Forms of Florpyrauxifen-Benzyl.

ProcellaCOR® (active ingredient [ai], florpyrauxifen-benzyl) is an aquatic herbicide registered for use in 2018 for managing invasive and nuisance macrophyte species. Registration studies evaluating its acute toxicity revealed a favorable environmental profile; however, prior to this study, no information existed on the toxicity of florpyrauxifen-benzyl to native freshwater mussels (Family Unionidae), one of the most sensitive and imperiled faunal groups globally. We followed standard acute (96h) toxicity test guidelines and exposed juvenile Fatmucket (Lampsilis siliquoidea) and Eastern Lampmussel (Lampsilis radiata) to the following formulations or compounds: ProcellaCOR SC and EC formulations, technical grade active ingredient (TGAI, florpyrauxifen-benzyl), and an analytical-grade sample of the weaker florpyrauxifen acid (FA). In all tests, the estimated median lethal concentrations to produce 50% mortality (LC50) were greater than the highest concentration tested of each formulation or compound. The no observable adverse effect concentrations (NOAEC, based on analytical recoveries measured at the highest concentration tested where no toxicity was observed) were TGAI = 26µg/L, FA = 100,000µg/L, ProcellaCOR® SC = 193µg ai/L ProcellaCOR® EC = 585µg ai/L and the NOAEC values for the registered commercial formulation products (ProcellaCOR® SC and ProcellaCOR® EC) were orders of magnitude greater (3.9× and 11.7×, respectively) than the maximum application rate (50µg/L). Our results show that the herbicide formulations and compounds tested were not acutely toxic to juveniles of these two species of freshwater mussels, indicating minimal risk of short-term exposure from florpyrauxifen-benzyl applications in the environment for aquatic weed control. However, potential chronic or sublethal effects remain uncharacterized and warrant additional investigation.

Comparison of Acute and Chronic Toxicity Laboratory Bioassay Endpoints with Benthic Community Responses in Field-Exposed Contaminated Sediments.

Sediment toxicity is usually assessed by conducting laboratory bioassays on thoroughly homogenized, field-collected, sediment samples. Although it is generally held that these bioassays provide a conservative assessment of the potential for environmental impact, there are few studies comparing the results of laboratory sediment bioassays with actual measures of benthic community health in exposed field populations. To help inform an understanding of the relative efficacy of laboratory-based bioassays in predicting potential impacts in exposed field populations, a laboratory-to-field comparison study was conducted. Laboratory bioassays included standard 10-d acute toxicity tests measuring survival in 4 species of estuarine/marine amphipods (Eohaustorius estuarius, Ampelisca abdita, Rhepoxinius abronius, and Leptocheirus plumulosus) and 2 longer term, 28-d sublethal tests with a marine polychaete, Neanthes arenaceodentata (survival and growth), and the amphipod L. plumulosus (survival, growth, and reproduction). A highly contaminated and toxic sediment was mixed with a cleaner sediment of similar grain size to produce a series of diluted contaminated sediment treatments (0, 6, 12, 25, and 50%). Sediment treatments were placed in containers and deployed in the field. At specified intervals (at time of deployment and 9 and 12 mo post deployment), containers were retrieved from the field and analyzed for sediment chemistry, infaunal community composition, and toxicity. Laboratory toxicity endpoints were compared with measures of benthic community health to evaluate the ability of the toxicity tests to accurately predict benthic impacts. The results of these comparisons indicate that the laboratory tests evaluated provide conservative estimates of potential benthic community impacts, with both acute and chronic tests detecting effects at lower treatment levels than were detected in exposed field populations using traditional measures of benthic community health. Environ Toxicol Chem 2019;38:1784-1802. Published 2019 Wiley Periodicals, Inc. on behalf of SETAC. This article is a US government work, and as such, is in the public domain in the United States of America.

Effects of silver and zinc on tropical freshwater organisms: Implications on water quality guidelines and ecological risk assessment

Tropical freshwater ecosystems comprise valuable habitats and important biodiversity. There is an imperative need to build a framework for establishing appropriate water quality guidelines of chemical contaminants in order to safeguard freshwater ecosystems in the tropics. Silver (Ag) and zinc (Zn) are known to be toxic to freshwater organisms at elevated concentrations. However, toxicity data of tropical freshwater species for these metals are lacking. To address this issue, we first conducted standard acute toxicity tests of each of the metal using eight tropical freshwater species based on measured concentrations in test solutions. Subsequently, we tested the null hypothesis that there are no differences in species sensitivities towards Ag and Zn between tropical and temperate species by comparing their corresponding species sensitivity distributions (SSDs). The results of SSD comparisons indicated that tropical species were more acutely sensitive to their temperate counterparts to Ag, while temperate species were slightly more sensitive towards Zn. Temperate-to-tropic extrapolation factor of 5 was recommended for Ag, while acute-to-chronic ratios (ACRs) of 100 and 10 were advocated for Ag and Zn, respectively. Through comparison of taxon-specific SSDs, we observed that crustaceans were the most sensitive taxon to Ag and Zn, and thus suitable for toxicity testing and risk assessment. Based on our results, concentrations of 0.004 and 6.4 μg L−1 (at hardness of 50 mg L−1 CaCO3) were recommended as interim chronic predicted no-effect concentrations for Ag and Zn, respectively for protecting freshwater ecosystems in tropical regions.

Variation in toxicity and ecological risks associated with some oil sands groundwaters

The surface mining of oil sands deposits requires the removal of groundwater to stabilize the deposit (depressurization) and make it safe for mining. The chemistry and toxicity of deep groundwaters (from 45 to 144 m below an active mining operation) were characterized to determine if the release of groundwaters would pose a risk to a receiving aquatic environment. Concentrations of conventional chemicals such as nutrients and metals were generally below CCME chronic guidelines. Concentrations of oil sands naphthenic acids (NAs) varied depending on the method of measurement and were routinely >1 mg L−1. Groundwaters rarely caused lethality to fish and invertebrates in standard acute and chronic toxicity tests. Algal cell production was negatively correlated with chlorides and potentially negatively with NAs. Other chronic toxicity variations were less obviously correlated with measured chemistry. The groundwaters had moderately-high oxygen demand (2 to 33 mg L−1), likely associated with nutrients and organic substances, and thus have the potential to enrich receiving surface water environments if left untreated and depending on the receiving environment. This paper presents for the first time a comprehensive (3 year) pairing of water chemistry and toxicity data on groundwaters collected from aquifer depressurization wells below an active oil sands operation. These data will contribute to a better understanding of the environmental risk these waters potentially pose, and ultimately, to the improvement of water management strategies and the reduction of the overall surface mining footprint of oil sands operations.

Petroleum hydrocarbon mixture toxicity and a trait-based approach to soil invertebrate species for site-specific risk assessments.

Although petroleum hydrocarbons released to the environment typically occur as mixtures, petroleum hydrocarbon remediation guidelines often reflect individual substance toxicity. It is well documented that groups of aliphatic petroleum hydrocarbons act via the same mechanism of action (nonpolar narcosis) and, theoretically, concentration addition mixture toxicity principles apply. To assess this theory, 10 standardized acute and chronic soil invertebrate toxicity tests on a range of organisms (Eisenia fetida, Lumbricus terrestris, Enchytraeus crypticus, Folsomia candida, Oppia nitens, and Hypoaspis aculeifer) were conducted with a refined petroleum hydrocarbon binary mixture. Reference models for concentration addition and independent action were applied to the mixture toxicity data with consideration of synergism, antagonism, and dose level toxicity. Both concentration addition and independent action, without further interactions, provided the best fit with observed response to the mixture. Individual fraction effective concentration values were predicted from optimized, fitted reference models. Concentration addition provided a better estimate than independent action of individual fraction effective concentrations based on comparison with available literature and species trends observed in toxic responses to the mixture. Interspecies differences in standardized laboratory soil invertebrate species responses to petroleum hydrocarbon-contaminated soil was reflected in unique traits. Diets that included soil, large body size, permeable cuticle, low lipid content, lack of ability to molt, and no maternal transfer were traits linked to a sensitive survival response to petroleum hydrocarbon-contaminated soil in laboratory tests. Traits linked to sensitive reproduction response in organisms tested were long life span and small clutch size. By deriving single-fraction toxicity endpoints considerate of mixtures, we can reduce the resources and time required to conduct site-specific risk assessments for the protection of a soil organism's exposure pathway. Environ Toxicol Chem 2018;37:2222-2234. © 2018 SETAC.

Advances in understanding the response of fish to linear alcohols in the environment

Short to long chain alcohols have a range of ecotoxicity to aquatic life driven by hydrophobic interactions with biological membranes. Carbon chain length and octanol:water partitioning coefficients are surrogates for hydrophobicity and strongly relate to aquatic toxicity. In these investigations, the toxicity of ethanol to 1-n-dodecanol to juvenile fish in standard acute toxicity tests is reviewed. Toxicity tests employing fish embryos (zebrafish Danio rerio and fathead minnow Pimephales promelas) in the Fish Embryo Test (OECD 236) format were conducted from C2 to C10 to compare against standard juvenile fish toxicity. Quantitative structure activity relationships for FET and fish individually and combined demonstrate that embryos are not different in sensitivity to juvenile fish. A combined QSAR was developed of the form Log 96 h LC50 (mM/L) = −0.925*log Kow + 2.060 (R2 10 = 0.954). Alcohols of 11–12 carbons show a deflection in the QSAR as toxicity approaches the solubility limit. Alcohols with longer chain lengths may only be tested at lower exposures relevant for chronic toxicity. Decanol was evaluated in a 33-d fish early life stage test (OECD 210) and survival was the most sensitive endpoint (EC10 = 0.43 mg/L, 0.0027 mM/L). This study suggests a reasonable acute to chronic ratio of 6.5 in line with historical literature for non-polar narcotic compounds. Fish are not uniquely more sensitive than Daphnia magna which suggests estimations of environmental hazard can be confidently made with either taxon. The overall environmental risk assessments for the longer chain alcohols included in this research remain largely unchanged primarily due to previous research demonstrating a very minimal environmental exposure even for highly toxic members of the category.

Applicability of the fish embryo acute toxicity (FET) test (OECD 236) in the regulatory context of Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH).

In 2013 the Organisation for Economic Co-operation and Development (OECD) test guideline (236) for fish embryo acute toxicity (FET) was adopted. It determines the acute toxicity of chemicals to embryonic fish. Previous studies show a good correlation of FET with the standard acute fish toxicity (AFT) test; however, the potential of the FET test to predict AFT, which is required by the Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH) regulation (EC 1907/2006) and the Classification, Labelling and Packaging (CLP) Regulation (EC 1272/2008), has not yet been fully clarified. In 2015 the European Chemicals Agency (ECHA) requested that a consultant perform a scientific analysis of the applicability of FET to predict AFT. The purpose was to compare the toxicity of substances to fish embryos and to adult fish, and to investigate whether certain factors (e.g., physicochemical properties, modes of action, or chemical structures) could be used to define the applicability boundaries of the FET test. Given the limited data availability, the analysis focused on organic substances. The present critical review summarizes the main findings and discusses regulatory application of the FET test under REACH. Given some limitations (e.g., neurotoxic mode of action) and/or remaining uncertainties (e.g., deviation of some narcotic substances), it has been found that the FET test alone is currently not sufficient to meet the essential information on AFT as required by the REACH regulation. However, the test may be used within weight-of-evidence approaches together with other independent, relevant, and reliable sources of information. The present review also discusses further research needs that may overcome the remaining uncertainties and help to increase acceptance of FET as a replacement for AFT in the future. For example, an increase in the availability of data generated according to OECD test guideline 236 may provide evidence of a higher predictive power of the test. Environ Toxicol Chem 2018;37:657-670. © 2017 SETAC.

Removal of chlorpyrifos insecticide in constructed wetlands with different plant species

ABSTRACT The objective of this study was to evaluate the remediation of water containing the insecticide chlorpyrifos by using constructed wetlands (CW) cultivated with Polygonum punctatum, Cynodon spp. and Mentha aquatica, operated under different hydraulic retention times: 24, 48, 96, 144 and 192 h. The system efficiency was based on reduction of the initial concentration of chlorpyrifos and toxicity of the contaminated water. The results showed that constructed wetlands are an excellent alternative for remediation of the insecticide chlorpyrifos in aqueous medium. It was observed that the average overall removal efficiency of the insecticide was 98.6%, and in the first hydraulic retention time, 24 h, chlorpyrifos was removed to levels below the detection limit in all CW. This result is mainly attributed to adsorption and microbial degradation. For the qualitative standard acute toxicity tests with Daphnia similis, for most samples there was a reduction in toxicity greater than 80%. It was reported that the ecotoxicological tests with the effluents of the constructed wetland are a good option as an indicator of the effectiveness of treatments and a promising alternative to complement the physical and chemical analyses.

Alternative acute oral toxicity assessment under REACH based on sub-acute toxicity values.

The REACH Regulation requires information on acute oral toxicity for substances produced or imported in quantities greater than one ton per year. When registering, animal testing should be used as last resort. The standard acute oral toxicity test requires use of animals. Therefore, the European Chemicals Agency examined whether alternative ways exist to generate information on acute oral toxicity. The starting hypothesis was that low acute oral toxicity can be predicted from the results of low toxicity in oral sub-acute toxicity studies. Proving this hypothesis would allow avoiding acute toxicity oral testing whenever a sub-acute oral toxicity study is required or available and indicates low toxicity. ECHA conducted an analysis of the REACH database and found suitable studies on both acute oral and sub-acute oral toxicities for 1,256 substances. 415 of these substances had low toxicity in the sub-acute toxicity study (i.e., NO(A)EL at or above the limit test threshold of 1,000 mg/kg). For 98% of these substances, low acute oral toxicity was also reported (i.e., LD50 above the classification threshold of 2,000 mg/kg). On the other hand, no correlation was found between lower NO(A)ELs and LD50. According to the REACH Regulation, this approach for predicting acute oral toxicity needs to be considered as part of a weight of evidence analysis. Therefore, additional sources of information to support this approach are presented. Ahead of the last REACH registration deadline, in 2018, ECHA estimates that registrants of about 550 substances can omit the in vivo acute oral toxicity study by using this adaptation.

Evaluation of the antiplasmodial properties of selected plants in southern Ethiopia.

BackgroundThe majority of the Ethiopian population is at risk of malaria largely caused by Plasmodium falciparum. The resistance of the parasite to existing drugs is the main challenge in the control of the disease and thus new therapeutic drugs are required. In Ethiopia, people use different plant species to treat malaria. However, very few of them have so far been evaluated for their safety level and antimalarial activity. Thus, the aim of this study was to evaluate the safety and antimalarial activity of extracts of Ajuga integrifolia, Clerodendrum myricoides, Melia azedarach, Peponium vogelii and Premna schimperi, locally used by the Sidama people of Ethiopia to treat malaria.MethodsThe safety level of 80 % methanol extracts of the plants were evaluated using standard acute toxicity test procedure. The antiplasmodial activity of 80 % methanol extracts of the plants were assessed in vivo using Swiss albino mice against chloroquine sensitive rodent malaria parasite, Plasmodium berghei, using the standard 4-day suppressive test procedure at doses of 200,400 and 800 mg/kg/day. The 80 % methanol extract of Ajuga integrifolia that exhibited better antimalarial activity was fractionated using different solvents and screened for its phytochemical constituents and evaluated in vivo for its antimalarial activity at doses of 100, 200 and 400 mg/kg/day.ResultsAll extracts given at the three different doses caused no lethal effect on mice in 24 h and within 10 days of observation. All extracts and fractions exhibited antimalarial activity in a dose dependant manner. The highest inhibition was exhibited by the crude extracts of A. integrifolia (35.17 %) at 800 mg/kg/day (P < 0.05). Among fractions of A. integrifolia, n-butanol fraction demonstrated the highest inhibition (29.80 %) at 400 mg/kg/day (P < 0.05). The extracts and fractions prolonged the survival time and prevented weight loss of the mice, but did not prevent PCV reduction. Phytochemical test on Ajuga integrifolia indicated the presence of alkaloids, flavonoids, saponins, terpenoids, anthraquinone, steroids, tannins, phenols and fatty acids.ConclusionsFindings show that the plants are non-toxic and demonstrate antimalarial activity in a dose dependant manner suporting claims of their traditional therapeutic value for malaria treatment. However, further in-depth investigation is required to assess the potential of the plants towards the development of new antimalarial agent.

Effects of unionised ammonia on tropical freshwater organisms: Implications on temperate-to-tropic extrapolation and water quality guidelines

Unionised ammonia (NH3) is highly toxic to freshwater organisms. Yet, most of the available toxicity data on NH3 were predominantly generated from temperate regions, while toxicity data on NH3 derived from tropical species were limited. To address this issue, we first conducted standard acute toxicity tests on NH3 using ten tropical freshwater species. Subsequently, we constructed a tropical species sensitivity distribution (SSD) using these newly generated toxicity data and available tropical toxicity data of NH3, which was then compared with the corresponding temperate SSD constructed from documented temperate acute toxicity data. Our results showed that tropical species were generally more sensitive to NH3 than their temperate counterparts. Based on the ratio between temperate and tropical hazardous concentration 10% values, we recommend an extrapolation factor of four to be applied when surrogate temperate toxicity data or temperate water quality guidelines of NH3 are used for protecting tropical freshwater ecosystems.

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.