Advancing botanical safety: A strategy for selecting, sourcing, and characterizing botanicals for developing toxicological tools.

Increases in botanical use, encompassing herbal medicines and dietary supplements, have underlined a critical need for an advancement in safety assessment methodologies. However, botanicals present unique challenges for safety assessment due to their complex and variable composition arising from diverse growing conditions, processing methods, and plant varieties. Historically, botanicals have been largely evaluated based on their history of use information, based primarily on traditional use or dietary history. However, this presumption lacks comprehensive toxicological evaluation, demanding innovative and consistent assessment strategies. To address these challenges, the Botanical Safety Consortium (BSC) was formed as an international, cross-sector forum of experts to identify fit-for purpose assays that can be used to evaluate botanical safety. This global effort aims to assess botanical safety assessment methodologies, merging traditional knowledge with modern in vitro and in silico assays. The ultimate goal is to champion the development of toxicity tools for botanicals. This manuscript highlights: 1) BSC's strategy for botanical selection, sourcing, and preparation of extracts to be used in in vitro assays, and 2) the approach utilized to characterize botanical extracts, using green tea and Asian ginseng as examples, to build confidence for use in biological assays.

A comparison of two methods for estimating critical swimming speed (Ucrit) in larval fathead minnows: the laminar flow assay and the spinning task assay.

Critical swimming speed (Ucrit) is considered a good predictor of swimming capabilities in fish. To estimate Ucrit, a fish is exposed to an incrementally increasing laminar flow of water until it cannot maintain its position against the current. The spinning task assay has been proposed as an alternative method to traditional laminar flow methods; however, these methods have not been directly compared. Thus, the goal of this study was to determine whether the spinning task assay is a suitable alternative to traditional laminar flow assays. To that end, the performance of fathead minnows in each assay was compared at three time points (14, 19 and 24 days post-fertilization, dpf). In 14 dpf fish, Ucrit estimates were similar regardless of the assay used. However, at 19 and 24 dpf, Ucrit estimates derived from the two assay types were significantly different. This indicates that the assays are not equivalent to one another and that the spinning task assay is not a suitable alternative to the laminar flow assay for the determination of Ucrit.

Investigating the Predictive Power of Three Potential Sublethal Endpoints for the Fathead Minnow Fish Embryo Toxicity Test: Snout-Vent Length, Eye Size, and Pericardial Edema.

The fish embryo acute toxicity (FET) test is known to be less sensitive than the fish acute test for some chemicals, including neurotoxicants. Thus, there is an interest in identifying additional endpoints that can improve FET test performance. The goal of this project was to advance alternative toxicity testing methods by determining whether select developmental abnormalities-snout-vent length, eye size, and pericardial area-are linked to adverse alterations in ecologically-relevant behaviors and delayed mortality. Fathead minnow (Pimephales promelas) FET tests were conducted with 3,4-dicholoroaniline, cadmium, and perfluorooctanesulfonic acid (PFOS) and developmental abnormalities were quantified. Surviving eleutheroembryos were reared in clean water to 14 days post fertilization (dpf), during which time behaviors and mortality were evaluated. None of the abnormalities evaluated were predictive of behavioral alterations; however, embryos with ≥14% reductions in length or ≥3.54-fold increases in pericardial area had an 80% chance of mortality by 14 dpf. When these abnormalities were used as markers of mortality, the LC50s for cadmium and PFOS were less than those calculated using only standardized FET test endpoints and similar to those obtained via larval fish tests, indicating that the snout-vent length and pericardial area warrant consideration as standard FET test endpoints.

Development of a larval fathead minnow optomotor response assay for assessing visual function.

The optomotor response (OMR) is a position stabilizing reflex to whole-field visual motion, demonstrated by fish instinctually moving to follow alternating black and white stripes. The OMR assay is used to assess the visual function of fish. While researchers have been using the OMR assay for years to evaluate the visual acuity of fish, no standardized methods exist. Therefore, the goal of the present study was to develop a standardized protocol for the measurement of OMR in larval fathead minnows, a common model species in ecotoxicology. Results indicate that OMR is a potential endpoint for the assessment of vision in larval fathead minnows and can be quantified as early as 9 dpf. When running the OMR assay, a 4-min acclimation period should be implemented. There did not appear to be a learning component to response latency; however, high variation in likely impeded the ability to detect significant differences.•Optomotor response of fathead minnow larvae can be quantified as early as 9 dpf.•When running the assay, a 4-min acclimation period should be implemented.•Due to high variation, response latency is not recommended for use as an endpoint.

Comparison of behavioral assays for assessing toxicant-induced alterations in neurological function in larval fathead minnows.

Neuroactive compounds are routinely detected in surface waters at concentrations that pose potential threats to wildlife. Exposure to neurotoxicants can adversely affect exposed organism by altering ecologically-important behaviors (e.g., feeding and predator response) that are likely to have important repercussions for populations. These compounds can elicit behavioral effects at concentrations lower than those that induce overt toxicity as indicated by mortality or decreased growth. Though a wide variety of methods have been employed to assess the behavior of early life stage fish, it is unclear which assays are best suited for identifying ecologically-relevant behavioral changes following exposures to neurotoxicants. The goal of the present study was to promote the use of behavioral assays for assessing the behavioral impacts of exposure to neurotoxic compounds by comparing the performance of different behavioral assays in larval fish. To achieve this goal, the sensitivity and practicality of three behavioral assays (i.e., feeding, optomotor response, and C-start assays) were compared in larval fathead minnows exposed to a known neurotoxicant, chlorpyrifos. There were significant alterations in the performance of fathead minnow larvae in all three behavioral assays in response to a 12-d embryo-larval exposure to chlorpyrifos. However, feeding and C-start were the most practical of the selected assays, as they took less time and allowed for larger samples sizes. Further work to standardize behavioral testing methods, and to link alterations to ecologically-relevant behaviors, will help promote the use of these assays when investigating the potential environmental impacts of neurotoxic compounds.

Identifying sublethal endpoints for evaluating neurotoxic compounds utilizing the fish embryo toxicity test.

Fish embryos are increasingly being utilized in aquatic toxicity testing, as evidenced by the Organisation for Economic Co-operation and Development's approval of the fish embryo acute toxicity (FET) test. However, the FET test only allows for the estimation of acute toxicity, whereas other test methods such as the larval growth and survival (LGS) test allow for the estimation of both acute and chronic toxicity. Additionally, it has been demonstrated that the FET test is less sensitive than other test methods for some neurotoxic compounds. To address these limitations, efforts to identify sublethal endpoints that increase FET test sensitivity and allow for the prediction of sublethal adverse effects have begun. As such, the objectives of the current study were 1) to compare estimated LC50 values from the FET and LGS test for three known neurotoxicants: fluoride (F), nickel (Ni), and cadmium (Cd) and 2) to evaluate the responsiveness of potential sublethal endpoints for the FET test related to growth (i.e., wet weight and snout-vent length), neurological development (i.e., spontaneous contraction frequency and eye size), and cardiovascular function (i.e., heart rate and pericardial area). The calculated LC50 values from the F and Cd FET test were significantly higher than those from the LGS test, demonstrating that the FET test is less sensitive than the LGS test for neurotoxic compounds. Only Cd exposure resulted in alterations in any of the sublethal endpoints investigated. Embryos/eleutheroembryos exposed to Cd displayed alterations in length, eye size, and pericardial area at concentrations five-fold less than the estimated LC50 value, suggesting that for Cd the inclusion of these sublethal endpoints would improve the sensitivity of the FET test. Overall, these results provide evidence that for some neurotoxicants, the inclusion of sublehtal endpoints may improve the utility of the FET test; however, further research utilizing a broader range of neurotoxicants with differing mechanisms of action, is needed to fully establish such endpoints in the context of routine FET test.

Development of cardiovascular and neurodevelopmental metrics as sublethal endpoints for the Fish embryo toxicity test.

The fathead minnow fish embryo toxicity (FET) test has been proposed as a more humane alternative to current toxicity testing methods as younger organisms are thought to experience less distress during toxicant exposure. However, the FET test protocol does not include endpoints that allow for the prediction of sublethal adverse outcomes, limiting its utility relative to other test types. Researchers have proposed the development of sublethal endpoints for the FET test to increase its utility. The present study 1) developed methods for previously unmeasured sublethal metrics in fathead minnows (i.e., spontaneous contraction frequency and heart rate) and 2) investigated the responsiveness of several sublethal endpoints related to growth (wet wt, length, and growth-related gene expression), neurodevelopment (spontaneous contraction frequency, eye size, and neurodevelopmental gene expression), and cardiovascular function and development (pericardial area, heart rate, and cardiovascular system-related gene expression) as additional FET test metrics using the model toxicant 3,4-dichloroaniline. Of the growth, neurological, and cardiovascular endpoints measured, length, eye size, and pericardial area were found to be more responsive than the other endpoints evaluated. Future studies linking alterations in these endpoints to longer-term adverse impacts are needed to fully evaluate the predictive power of these metrics in chemical and whole-effluent toxicity testing. Environ Toxicol Chem 2018;37:2530-2541. © 2018 SETAC.

Enhancing the fathead minnow fish embryo toxicity test: Optimizing embryo production and assessing the utility of additional test endpoints.

The fathead minnow fish embryo toxicity (FET) test has been identified as a potential alternative to toxicity test methods that utilize older fish. However, several challenges have been identified with the fathead minnow FET test, including: 1) difficulties in obtaining appropriately-staged embryos for FET test initiation, 2) a paucity of data comparing fathead minnow FET test performance to the fathead minnow larval growth and survival (LGS) test and 3) a lack of sublethal endpoints that could be used to estimate chronic toxicity and/or predict adverse effects. These challenges were addressed through three study objectives. The first objective was to optimize embryo production by assessing the effect of breeding group composition (number of males and females) on egg production. Results showed that groups containing one male and four females produced the largest clutches, enhancing the likelihood of procuring sufficient numbers of embryos for FET test initiation. The second study objective was to compare the performance of the FET test to that of the fathead minnow LGS test using three reference toxicants. The FET and LGS tests were similar in their ability to predict the acute toxicity of sodium chloride and ethanol, but the FET test was found to be more sensitive than the LGS test for sodium dodecyl sulfate. The last objective of the study was to evaluate the utility and practicality of several sublethal metrics (i.e., growth, developmental abnormalities and growth- and stress-related gene expression) as FET test endpoints. Developmental abnormalities, including pericardial edema and hatch success, were found to offer the most promise as additional FET test endpoints, given their responsiveness, potential for predicting adverse effects, ease of assessment and low cost of measurement.

Photoenhanced Toxicity of Weathered Crude Oil in Sediment and Water to Larval Zebrafish.

Solar radiation exposure can increase the toxicity of bioaccumulated oil compounds in a diversity of aquatic species. We investigated the photoenhanced toxicity of weathered South Louisiana crude oil in sediment and water accommodated fractions (WAF) to larval zebrafish. Larvae were first exposed for 24 h to one of six treatments: no oil (sediment or water), 7.5 g oil/kg sediment, oil-only WAF, oil WAF plus the dispersant Corexit 9500A, or dispersant alone. Larvae were then exposed to high or low levels of sunlight in control water for 3 or 3.5 h. Hydrocarbon concentrations were measured in exposure media, including alkanes, polycyclic aromatic compounds and total petroleum hydrocarbons. Significant phototoxicity was observed in larvae exposed to oiled sediment, oil-only WAF, and oil plus dispersant WAF. The results indicated that petroleum from the northern Gulf of Mexico can be phototoxic to larval fish exposed to oil in either the water column or sediment.

Effects of Louisiana crude oil on the sheepshead minnow (Cyprinodon variegatus) during a life-cycle exposure to laboratory oiled sediment.

Determining the long-term effects of crude oil exposure is critical for ascertaining population-level ecological risks of spill events. A 19-week complete life-cycle experiment was conducted with the estuarine sheepshead minnow (Cyprinodon variegatus) exposed to reference (uncontaminated) sediment spiked with laboratory weathered South Louisiana crude (SLC) oil at five concentrations as well as one unspiked sediment control and one seawater (no sediment) control. Newly hatched larvae were exposed to the oiled sediments at measured concentrations of < 1 (sediment control), 50, 103, 193, 347, and 711 mg total polyaromatic hydrocarbons (tPAH)/kg dry sediment. Juveniles were exposed through the reproductively active adult phase at measured concentrations of <1 (sediment control), 52, 109, 199, 358, and 751 mg tPAH/kg sediment. Throughout the exposure, fish were assessed for growth, survival, and reproduction. Resulting F1 embryos were then collected, incubated, and hatched in clean water to determine if parental full life-cycle exposure to oiled sediment produced trans-generational effects. Larvae experienced significantly reduced standard length (5-13% reduction) and wet weight (13-35% reduction) at concentrations at and above 50 and 103 mg tPAH/kg sediment, respectively. At 92 and 132 days post hatch (dph), standard length was reduced (7-13% reduction) at 199 and 109 mg tPAH/kg dry sediment, respectively, and wet weight for both time periods was reduced at concentrations at and above 109 mg tPAH/kg dry sediment (21-38% reduction). A significant reduction (51-65%) in F0 fecundity occurred at the two highest test concentrations, but no difference was observed in F1 embryo survival. This study is the first to report the effects of chronic laboratory exposure to oiled sediment, and will assist the development of population models for evaluating risk to benthic spawning fish species exposed to oiled sediments. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1627-1639, 2016.

Developmental toxicity of Louisiana crude oil-spiked sediment to zebrafish.

Embryonic exposures to the components of petroleum, including polycyclic aromatic hydrocarbons (PAHs), cause a characteristic suite of developmental defects and cardiotoxicity in a variety of fish species. We exposed zebrafish embryos to reference sediment mixed with laboratory weathered South Louisiana crude oil and to sediment collected from an oiled site in Barataria Bay, Louisiana in December 2010. Laboratory oiled sediment exposures caused a reproducible set of developmental malformations in zebrafish embryos including yolk sac and pericardial edema, craniofacial and spinal defects, and tissue degeneration. Dose-response studies with spiked sediment showed that total polycyclic aromatic hydrocarbons (tPAH) concentrations of 27mg tPAH/kg (dry weight normalized to 1 percent organic carbon [1 percent OC]) caused a significant increase in defects, and concentrations above 78mg tPAH/kg 1 percent OC caused nearly complete embryo mortality. No toxicity was observed in Barataria sediment with 2mg tPAH/kg 1 percent OC. Laboratory aging of spiked sediment at 4°C resulted in a nearly 10-fold decrease in sensitivity over a 40-day period. This study demonstrates oiled sediment as an exposure pathway to fish with dose-dependent effects on embryogenesis that are consistent with PAH mechanisms of developmental toxicity. The results have implications for effects on estuarine fish from oiled coastal areas during the Deepwater Horizon spill.