Application of Effect-Based Methods to Water Quality Monitoring: Answering Frequently Asked Questions by Water Quality Managers, Regulators, and Policy Makers.

Effect-based methods (EBM) have great potential for water quality monitoring as they can detect the mixture effects of all active known and unknown chemicals in a sample, which cannot be addressed by chemical analysis alone. To date, EBM have primarily been applied in a research context, with a lower level of uptake by the water sector and regulators. This is partly due to concerns regarding the reliability and interpretation of EBM. Using evidence from the peer-reviewed literature, this work aims to answer frequently asked questions about EBM. The questions were identified through consultation with the water industry and regulators and cover topics related to the basis for using EBM, practical considerations regarding reliability, sampling for EBM and quality control, and what to do with the information provided by EBM. The information provided in this work aims to give confidence to regulators and the water sector to stimulate the application of EBM for water quality monitoring.

Effects of neonatal exposure to the flame retardant tetrabromobisphenol-A, aluminum diethylphosphinate or zinc stannate on long-term potentiation and synaptic protein levels in mice.

Brominated flame retardants such as tetrabromobisphenol-A (TBBPA) may exert (developmental) neurotoxic effects. However, data on (neuro)toxicity of halogen-free flame retardants (HFFRs) are scarce. Recent in vitro studies indicated a high neurotoxic potential for some HFFRs, e.g., zinc stannate (ZS), whereas the neurotoxic potential of other HFFRs, such as aluminum diethylphosphinate (Alpi), appears low. However, the in vivo (neuro)toxicity of these compounds is largely unknown. We therefore investigated effects of neonatal exposure to TBBPA, Alpi or ZS on synaptic plasticity in mouse hippocampus. Male C57bl/6 mice received a single oral dose of 211 µmol/kg bw TBBPA, Alpi or ZS on postnatal day (PND) 10. On PND 17-19, effects on hippocampal synaptic plasticity were investigated using ex vivo extracellular field recordings. Additionally, we measured levels of postsynaptic proteins involved in long-term potentiation (LTP) as well as flame retardant concentrations in brain, muscle and liver tissues. All three flame retardants induced minor, but insignificant, effects on LTP. Additionally, TBBPA induced a minor decrease in post-tetanic potentiation. Despite these minor effects, expression of selected synaptic proteins involved in LTP was not affected. The flame retardants could not be measured in significant amounts in the brains, suggesting low bioavailability and/or rapid elimination/metabolism. We therefore conclude that a single neonatal exposure on PND 10 to TBBPA, Alpi or ZS does affect neurodevelopment and synaptic plasticity only to a small extent in mice. Additional data, in particular on persistence, bioaccumulation and (in vivo) toxicity, following prolonged (developmental) exposure are required for further (human) risk assessment.

BDE-47 and 6-OH-BDE-47 modulate calcium homeostasis in primary fetal human neural progenitor cells via ryanodine receptor-independent mechanisms.

Polybrominated diphenyl ethers (PBDEs) are bioaccumulating flame retardants found in rising concentrations in human tissue. Epidemiological and animal studies have raised concern for their potential to induce developmental neurotoxicity (DNT). Considering the essential role of calcium homeostasis in neurodevelopment, PBDE-induced disturbance of intracellular calcium concentration ([Ca(2+)]i) may underlie PBDE-induced DNT. To test this hypothesis, we investigated acute effects of BDE-47 and 6-OH-BDE-47 on [Ca(2+)]i in human neural progenitor cells (hNPCs) and unraveled involved signaling pathways. Short-time differentiated hNPCs were exposed to BDE-47, 6-OH-BDE-47, and multiple inhibitors/stimulators of presumably involved signaling pathways to determine possible effects on [Ca(2+)]i by single-cell microscopy with the fluorescent dye Fura-2. Initial characterization of calcium signaling pathways confirmed the early developmental stage of hNPCs. In these cells, BDE-47 (2 µM) and 6-OH-BDE-47 (0.2 µM) induce [Ca(2+)]i transients. This increase in [Ca(2+)]i is due to extracellular Ca(2+) influx and intracellular release of Ca(2+), mainly from the endoplasmic reticulum (ER). While extracellular Ca(2+) seems to enter the cytoplasm upon 6-OH-BDE-47 by interfering with the cell membrane and independent of Ca(2+) ion channels, ER-derived Ca(2+) is released following activation of protein lipase C and inositol 1,4,5-trisphosphate receptor, but independently of ryanodine receptors. These findings illustrate that immature developing hNPCs respond to low concentrations of 6-OH-BDE-47 by an increase in [Ca(2+)]i and provide new mechanistic explanations for such BDE-induced calcium disruption. Thus, these data support the possibility of a critical window of PBDE exposure, i.e., early human brain development, which has to be acknowledged in risk assessment.

Making waves: Xanthates on the radar - Environmental risks and water quality impact.

Xanthates, derivatives of xanthic acid, are widely utilized across industries such as agrochemicals, rubber processing, pharmaceuticals, metallurgical, paper and mining to help separate metals from ore. Despite their prevalent use, many registered xanthates lack comprehensive information on potential risks to human health and the environment. The mining sector, a significant consumer of xanthates, drives demand. However, emissions into the environment remain poorly understood, especially concerning water quality. A recent EU parliamentary voting on water legislation highlights the urgency to address water pollution and the potential toxicity of xanthates. While limited data exist on xanthate presence in the environment, existing studies indicate their toxicity and contribution to environmental pollution, primarily due to carbon disulfide, a decomposition product. Concerns are mounting over the release of xanthates and carbon disulfide, particularly in mining areas near populated regions and river tributaries, raising questions about downstream impacts and public health risks. Proposed expansions of xanthate-reliant mining activities in Europe, heighten concerns about emissions and water quality impacts. Current databases lack xanthate-related monitoring data, hindering environmental and health risk assessments. Addressing this gap requires water sampling and chemical analysis and investigations into the use, occurrence, and potential impacts of xanthates from industrial activities on water bodies, including those used for drinking water production is imperative.

(Eco)toxicological tests for assessing impacts of chemical stress to aquatic ecosystems: Facts, challenges, and future.

Monitoring of chemicals in the aquatic environment by chemical analysis alone cannot completely assess and predict the effects of chemicals on aquatic species and ecosystems. This is primarily because of the increasing number of (unknown) chemical stressors and mixture effects present in the environment. In addition, the ability of ecological indices to identify underlying stressors causing negative ecological effects is limited. Therefore, additional complementary methods are needed that can address the biological effects in a direct manner and provide a link to chemical exposure, i.e. (eco)toxicological tests. (Eco)toxicological tests are defined as test systems that expose biological components (cells, individuals, populations, communities) to (environmental mixtures of) chemicals to register biological effects. These tests measure responses at the sub-organismal (biomarkers and in vitro bioassays), whole-organismal, population, or community level. We performed a literature search to obtain a state-of-the-art overview of ecotoxicological tests available for assessing impacts of chemicals to aquatic biota and to reveal datagaps. In total, we included 509 biomarkers, 207 in vitro bioassays, 422 tests measuring biological effects at the whole-organismal level, and 78 tests at the population- community- and ecosystem-level. Tests at the whole-organismal level and biomarkers were most abundant for invertebrates and fish, whilst in vitro bioassays are mostly based on mammalian cell lines. Tests at the community- and ecosystem-level were almost missing for organisms other than microorganisms and algae. In addition, we provide an overview of the various extrapolation challenges faced in using data from these tests and suggest some forward looking perspectives. Although extrapolating the measured responses to relevant protection goals remains challenging, the combination of ecotoxicological experiments and models is key for a more comprehensive assessment of the effects of chemical stressors to aquatic ecosystems.

Development of a framework to derive effect-based trigger values to interpret CALUX data for drinking water quality.

Bioassays are increasingly being implemented for water quality monitoring as targeted chemical analyses are not always sufficient for the detection of all emerging chemicals or transformation products. However, the interpretation of bioassay results remains challenging, in particular because a positive response does not necessarily indicate that there may be an increased risk. For this purpose, effect-based trigger (EBT) values have been introduced as thresholds above which action needs to be undertaken to determine the cause of the response. The goals of this study were to (i) evaluate various approaches used to determine EBT values and (ii) based on the findings, derive human health EBT values for Chemical Activated LUciferase gene eXpression (CALUX) in vitro bioassays used for routine monitoring of water quality in the Netherlands. Finally, (iii) an uncertainty analysis was carried out to determine the protective power of the derived EBT values and the chance that potentially harmful substances might not be detected. EBT values that can be implemented in routine monitoring could be determined for four of eight selected bioassays. These EBT were compared to bioassay results from routine water quality monitoring carried out in the Netherlands. Furthermore, a framework for the calculation and evaluation of derived EBT values for routine application to monitor drinking water and its sources is proposed.

Comparing conventional and green fracturing fluids by chemical characterisation and effect-based screening.

There is public and scientific concern about air, soil and water contamination and possible adverse environmental and human health effects as a result of hydraulic fracturing activities. The use of greener chemicals in fracturing fluid aims to mitigate these effects. This study compares fracturing fluids marketed as either 'conventional' or 'green', as assessed by their chemical composition and their toxicity in bioassays. Chemical composition was analysed via non-target screening using liquid chromatography - high resolution mass spectrometry, while toxicity was evaluated by the Ames fluctuation test to assess mutagenicity and CALUX reporter gene assays to determine specific toxicity. Overall, the results do not indicate that the 'green' fluids are less harmful than the 'conventional' ones. First, there is no clear indication that the selected green fluids contain chemicals present at lower concentrations than the selected conventional fluids. Second, the predicted environmental fate of the identified compounds does not seem to be clearly distinct between the 'green' and 'conventional' fluids, based on the available data for the top five chemicals based on signal intensity that were tentatively identified. Furthermore, Ames fluctuation test results indicate that the green fluids have a similar genotoxic potential than the conventional fluids. Results of the CALUX reporter gene assays add to the evidence that there is no clear difference between the green and conventional fluids. These results do not support the claim that currently available and tested green-labeled fracturing fluids are environmentally more friendly alternatives to conventional fracturing fluids.

Development of a quantitative chemical risk assessment (QCRA) procedure for contaminants of emerging concern in drinking water supply.

The uncertainties on the occurrence, fate and hazard of Contaminants of Emerging Concern (CECs) increasingly challenge drinking water (DW) utilities whether additional measures should be taken to reduce the health risk. This has led to the development and evaluation of risk-based approaches by the scientific community. DW guideline values are commonly derived based on deterministic chemical risk assessment (CRA). Here, we propose a new probabilistic procedure, that is a quantitative chemical risk assessment (QCRA), to assess potential health risk related to the occurrence of CECs in DW. The QCRA includes uncertainties in risk calculation in both exposure and hazard assessments. To quantify the health risk in terms of the benchmark quotient probabilistic distribution, the QCRA estimates the probabilistic distribution of CECs concentration in DW based on their concentration in source water and simulating the breakthrough curves of a granular activated carbon (GAC) treatment process. The model inputs and output uncertainties were evaluated by sensitivity and uncertainty analyses for each step of the risk assessment to identify the most relevant factors affecting risk estimation. Dominant factors resulted to be the concentration of CECs in water sources, GAC isotherm parameters and toxicological data. To stress the potential of this new QCRA approach, several case studies are considered with focus on bisphenol A as an example CEC and various GAC management options. QCRA quantifies the probabilistic risk, providing more insight compared to CRA. QCRA proved to be more effective in supporting the intervention prioritization for treatment optimization to pursue health risk minimization.

Integration of target analyses, non-target screening and effect-based monitoring to assess OMP related water quality changes in drinking water treatment.

The ever-increasing production and use of chemicals lead to the occurrence of organic micro-pollutants (OMPs) in drinking water sources, and consequently the need for their removal during drinking water treatment. Due to the sheer number of OMPs, monitoring using targeted chemical analyses alone is not sufficient to assess drinking water quality as well as changes thereof during treatment. High-resolution mass spectrometry (HRMS) based non-target screening (NTS) as well as effect-based monitoring using bioassays are promising monitoring tools for a more complete assessment of water quality and treatment performance. Here, we developed a strategy that integrates data from chemical target analyses, NTS and bioassays. We applied it to the assessment of OMP related water quality changes at three drinking water treatment pilot installations. These installations included advanced oxidation processes, ultrafiltration in combination with reverse osmosis, and granular activated carbon filtration. OMPs relevant for the drinking water sector were spiked into the water treated in these installations. Target analyses, NTS and bioassays were performed on samples from all three installations. The NTS data was screened for predicted and known transformation products of the spike-in compounds. In parallel, trend profiles of NTS features were evaluated using multivariate analysis methods. Through integration of the chemical data with the biological effect-based results potential toxicity was accounted for during prioritization. Together, the synergy of the three analytical methods allowed the monitoring of OMPs and transformation products, as well as the integrative biological effects of the mixture of chemicals. Through efficient analysis, visualization and interpretation of complex data, the developed strategy enabled to assess water quality and the impact of water treatment from multiple perspectives. Such information could not be obtained by any of the three methods alone. The developed strategy thereby provides drinking water companies with an integrative tool for comprehensive water quality assessment.

Prioritizing anthropogenic chemicals in drinking water and sources through combined use of mass spectrometry and ToxCast toxicity data.

Advancements in high-resolution mass spectrometry based methods have enabled a shift from pure target analysis to target, suspect and non-target screening analyses to detect chemicals in water samples. The multitude of suspect chemicals thereby detected needs to be prioritized for further identification, prior to health risk assessment and potential inclusion into monitoring programs. Here, we compare prioritization of chemicals in Dutch water samples based on relative intensities only to prioritization including hazard information based on high-throughput in vitro toxicity data. Over 1000 suspects detected in sewage treatment plant effluent, surface water, groundwater and drinking water samples were ranked based on their relative intensities. Toxicity data availability and density in the ToxCast database were determined and visualized for these suspects, also in regard to water relevant mechanisms of toxicity. More than 500 suspects could be ranked using occurrence/hazard ratios based on more than 1000 different assay endpoints. The comparison showed that different prioritization strategies resulted in significantly different ranking, with only 2 suspects prioritized based on occurrence among the top 20 in the hazard ranking. We therefore propose a novel scheme that integrates both exposure and hazard data, and efficiently prioritizes which features need to be confidently identified first.

Hydroxylation increases the neurotoxic potential of BDE-47 to affect exocytosis and calcium homeostasis in PC12 cells.

BACKGROUND: Oxidative metabolism, resulting in the formation of hydroxylated polybrominated diphenyl ether (PBDE) metabolites, may enhance the neurotoxic potential of brominated flame retardants. OBJECTIVE: Our objective was to investigate the effects of a hydroxylated metabolite of 2,2',4,4'-tetra-bromodiphenyl ether (BDE-47; 6-OH-BDE-47) on changes in the intracellular Ca2+ concentration ([Ca2+]i) and vesicular catecholamine release in PC12 cells. METHODS: We measured vesicular catecholamine release and [Ca2+]i using amperometry and imaging of the fluorescent Ca2+-sensitive dye Fura-2, respectively. RESULTS: Acute exposure of PC12 cells to 6-OH-BDE-47 (5 microM) induced vesicular catecholamine release. Catecholamine release coincided with a transient increase in [Ca2+]i, which was observed shortly after the onset of exposure to 6-OH-BDE-47 (120 microM). An additional late increase in [Ca2+]i was often observed at > or =1 microM 6-OH-BDE-47. The initial transient increase was absent in cells exposed to the parent compound BDE-47, whereas the late increase was observed only at 20 microM. Using the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and thapsigargin to empty intracellular Ca2+ stores, we found that the initial increase originates from emptying of the endoplasmic reticulum and consequent influx of extracellular Ca2+, whereas the late increase originates primarily from mitochondria. CONCLUSION: The hydroxylated metabolite 6-OH-BDE-47 is more potent in disturbing Ca2+ homeostasis and neurotransmitter release than the parent compound BDE-47. The present findings indicate that bioactivation by oxidative metabolism adds considerably to the neurotoxic potential of PBDEs. Additionally, based on the observed mechanism of action, a cumulative neurotoxic effect of PBDEs and ortho-substituted polychlorinated biphenyls on [Ca2+]i cannot be ruled out.

Exploration of ToxCast/Tox21 bioassays as candidate bioanalytical tools for measuring groups of chemicals in water.

The present study explores the ToxCast/Tox21 database to select candidate bioassays as bioanalytical tools for measuring groups of chemicals in water. To this aim, the ToxCast/Tox21 database was explored for bioassays that detect polycyclic aromatic hydrocarbons (PAHs), aromatic amines (AAs), (chloro)phenols ((C)Ps) and halogenated aliphatic hydrocarbons (HAliHs), which are included in the European and/or Dutch Drinking Water Directives. Based on the analysis of the availability and performance of bioassays included in the database, we concluded that several bioassays are suitable as bioanalytical tools for assessing the presence of PAHs and (C)Ps in drinking water sources. No bioassays were identified for AAs and HAliHs, due to the limited activity of these chemicals and/or the limited amount of data on these chemicals in the database. A series of bioassays was selected that measure molecular or cellular effects that are covered by bioassays currently in use for chemical water quality monitoring. Interestingly, also bioassays were selected that represent molecular or cellular effects that are not covered by bioassays currently applied. The usefulness of these newly identified bioassays as bioanalytical tools should be further evaluated in follow-up studies. Altogether, this study shows how exploration of the ToxCast/Tox21 database provides a series of candidate bioassays as bioanalytical tools for measuring groups of chemicals in water. This assessment can be performed for any group of chemicals of interest (if represented in the database), and may provide candidate bioassays that can be used to complement the currently applied bioassays for chemical water quality assessment.

Neonatal exposure to brominated flame retardant BDE-47 reduces long-term potentiation and postsynaptic protein levels in mouse hippocampus.

BACKGROUND: Increasing environmental levels of brominated flame retardants raise concern about possible adverse effects, particularly through early developmental exposure. OBJECTIVE: The objective of this research was to investigate neurodevelopmental mechanisms underlying previously observed behavioral impairments observed after neonatal exposure to polybrominated diphenyl ethers (PBDEs). METHODS: C57Bl/6 mice received a single oral dose of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) on postnatal day (PND) 10 (i.e., during the brain growth spurt). On PND17-19, effects on synaptic plasticity, levels of postsynaptic proteins involved in long-term potentiation (LTP), and vesicular release mechanisms were studied ex vivo. We investigated possible acute in vitro effects of BDE-47 on vesicular catecholamine release and intracellular Ca(2+) in rat pheochromocytoma (PC12) cells. RESULTS: Field-excitatory postsynaptic potential (f-EPSP) recordings in the hippocampal CA1 area demonstrated reduced LTP after exposure to 6.8 mg (14 micromol)/kg body weight (bw) BDE-47, whereas paired-pulse facilitation was not affected. Western blotting of proteins in the postsynaptic, triton-insoluble fraction of hippocampal tissue revealed a reduction of glutamate receptor subunits NR2B and GluR1 and autophosphorylated-active Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII), whereas other proteins tested appeared unaffected. Amperometric recordings in chromaffin cells from mice exposed to 68 mg (140 micromol)/kg bw BDE-47 did not reveal changes in catecholamine release parameters. Modest effects on vesicular release and intracellular Ca(2+) in PC12 cells were seen following acute exposure to 20 microM BDE-47. The combined results suggest a post-synaptic mechanism in vivo. CONCLUSION: Early neonatal exposure to a single high dose of BDE-47 causes a reduction of LTP together with changes in postsynaptic proteins involved in synaptic plasticity in the mouse hippocampus.

Mechanisms of Action Point Towards Combined PBDE/NDL-PCB Risk Assessment.

At present, human risk assessment of the structurally similar non-dioxin-like (NDL) PCBs and polybrominated diphenylethers (PBDEs) is done independently for both groups of compounds. There are however obvious similarities between NDL-PCBs and PBDEs with regard to modulation of the intracellular calcium homeostasis (basal calcium levels, voltage-gated calcium channels, calcium uptake, ryanodine receptor) and thyroid hormone (TH) homeostasis (TH levels and transport). which are mechanisms of action related to neurobehavioral effects (spontaneous activity, habituation and learning ability). There also similarities in agonistic interactions with the hepatic nuclear receptors PXR and CAR. Several effects on developmental (reproductive) processes have also been observed, but results were more dispersed and insufficient to compare both groups of compounds. The available mechanistic information is sufficient to warrant a dose addition model for NDL-PCBs and PBDEs, including their hydroxylated metabolites.Although many of the observed effects are similar from a qualitative point of view for both groups, congener or tissue specific differences have also been found. As this is a source of uncertainty in the combined hazard and risk assessment of these compounds, molecular entities involved in the observed mechanisms and adverse outcomes associated with these compounds need to be identified. The systematical generation of (quantitative) structure-activity information for NDL-PCBs and PBDEs on these targets (including potential non-additive effects) will allow a more realistic risk estimation associated with combined exposure to both groups of compounds during early life. Additional validation studies are needed to quantify these uncertainties for risk assessment of NDL-PCBs and PBDEs.

Chronic 14-day exposure to insecticides or methylmercury modulates neuronal activity in primary rat cortical cultures.

There is an increasing demand for in vitro test systems to detect neurotoxicity for use in chemical risk assessment. In this study, we evaluated the applicability of rat primary cortical cultures grown on multi-well micro-electrode arrays (mwMEAs) to detect effects of chronic 14-day exposure to structurally different insecticides or methylmercury on neuronal activity (mean spike rate; MSR). Effects of chronic exposure to α-cypermethrin, endosulfan, carbaryl, chlorpyrifos(-oxon), methylmercury or solvent control [14days exposure, initiated after baseline recording at day in vitro (DIV)7] were studied in five successive recordings between DIV10 and DIV21. The results were compared to effects of acute exposure to these same compounds (activity recorded immediately after the start of exposure after baseline recording at DIV10-11). Chronic 14-day exposure to methylmercury, chlorpyrifos and α-cypermethrin inhibited MSR, all with a lowest-observed effect concentration (LOEC) of 0.1µM, while exposure to endosulfan increased MSR [LOEC: 1µM]. No significant effects were observed for chlorpyrifos-oxon and carbaryl. Similar to the observations in the chronic 14-day exposure studies, MSR was inhibited by acute 30-min exposure to methylmercury, chlorpyrifos, and α-cypermethrin [LOECs: 1µM, 10µM, and 1µM, respectively], whereas endosulfan increased MSR [LOEC: 0.3µM]. While not observed in the chronic 14-day exposure study, acute exposure to chlorpyrifos-oxon and carbaryl resulted in inhibition of MSR [LOECs: 10µM, and100 µM, respectively]. Effects on median interspike intervals (mISI; a measure for neuronal firing pattern) were not detected following chronic 14-day or acute 30-min exposure, except for increased mISI at acute chlorpyrifos and α-cypermethrin exposures at concentrations that also inhibited MSR. These data indicate that the effects of chronic 14-day exposures to methylmercury and insecticides at low concentrations on spontaneous neuronal activity in vitro can be predicted in rapid acute screening studies using mwMEAs.

Inhibition of Voltage-Gated Calcium Channels After Subchronic and Repeated Exposure of PC12 Cells to Different Classes of Insecticides.

We previously demonstrated that acute inhibition of voltage-gated calcium channels (VGCCs) is a common mode of action for (sub)micromolar concentrations of chemicals, including insecticides. However, because human exposure to chemicals is usually chronic and repeated, we investigated if selected insecticides from different chemical classes (organochlorines, organophosphates, pyrethroids, carbamates, and neonicotinoids) also disturb calcium homeostasis after subchronic (24 h) exposure and after a subsequent (repeated) acute exposure. Effects on calcium homeostasis were investigated with single-cell fluorescence (Fura-2) imaging of PC12 cells. Cells were depolarized with high-K(+) saline to study effects of subchronic or repeated exposure on VGCC-mediated Ca(2+) influx. The results demonstrate that except for carbaryl and imidacloprid, all selected insecticides inhibited depolarization (K(+))-evoked Ca(2+) influx after subchronic exposure (IC50's: approximately 1-10 µM) in PC12 cells. These inhibitory effects were not or only slowly reversible. Moreover, repeated exposure augmented the inhibition of the K(+)-evoked increase in intracellular calcium concentration induced by subchronic exposure to cypermethrin, chlorpyrifos, chlorpyrifos-oxon, and endosulfan (IC50's: approximately 0.1-4 µM). In rat primary cortical cultures, acute and repeated chlorpyrifos exposure also augmented inhibition of VGCCs compared with subchronic exposure. In conclusion, compared with subchronic exposure, repeated exposure increases the potency of insecticides to inhibit VGCCs. However, the potency of insecticides to inhibit VGCCs upon repeated exposure was comparable with the inhibition previously observed following acute exposure, with the exception of chlorpyrifos. The data suggest that an acute exposure paradigm is sufficient for screening chemicals for effects on VGCCs and that PC12 cells are a sensitive model for detection of effects on VGCCs.

Inhibition of voltage-gated calcium channels as common mode of action for (mixtures of) distinct classes of insecticides.

Humans are exposed to distinct structural classes of insecticides with different neurotoxic modes of action. Because calcium homeostasis is essential for proper neuronal function and development, we investigated the effects of insecticides from different classes (pyrethroid: (α-)cypermethrin; organophosphate: chlorpyrifos; organochlorine: endosulfan; neonicotinoid: imidacloprid) and mixtures thereof on the intracellular calcium concentration ([Ca(2+)]i). Effects of acute (20 min) exposure to (mixtures of) insecticides on basal and depolarization-evoked [Ca(2+)]i were studied in vitro with Fura-2-loaded PC12 cells and high resolution single-cell fluorescence microscopy. The data demonstrate that cypermethrin, α-cypermethrin, endosulfan, and chlorpyrifos concentration-dependently decreased depolarization-evoked [Ca(2+)]i, with 50% (IC50) at 78nM, 239nM, 250nM, and 899nM, respectively. Additionally, acute exposure to chlorpyrifos or endosulfan (10µM) induced a modest increase in basal [Ca(2+)]i, amounting to 68 ± 8nM and 53 ± 8nM, respectively. Imidacloprid did not disturb basal or depolarization-evoked [Ca(2+)]i at 10µM. Following exposure to binary mixtures, effects on depolarization-evoked [Ca(2+)]i were within the expected effect additivity range, whereas the effect of the tertiary mixture was less than this expected additivity effect range. These results demonstrate that different types of insecticides inhibit depolarization-evoked [Ca(2+)]i in PC12 cells by inhibiting voltage-gated calcium channels (VGCCs) in vitro at concentrations comparable with human occupational exposure levels. Moreover, the effective concentrations in this study are below those for earlier described modes of action. Because inhibition of VGCCs appears to be a common and potentially additive mode of action of several classes of insecticides, this target should be considered in neurotoxicity risk assessment studies.

Characterization of calcium responses and electrical activity in differentiating mouse neural progenitor cells in vitro.

In vitro methods for developmental neurotoxicity (DNT) testing have the potential to reduce animal use and increase insight into cellular and molecular mechanisms underlying chemical-induced alterations in the development of functional neuronal networks. Mouse neural progenitor cells (mNPCs) differentiate into nervous system-specific cell types and have proven valuable to detect DNT using biochemical and morphological techniques. We therefore investigated a number of functional neuronal parameters in primary mNPCs to explore their applicability for neurophysiological in vitro DNT testing. Immunocytochemistry confirmed that mNPCs express neuronal, glial, and progenitor markers at various differentiation durations (1, 7, 14, and 21 days). Because intracellular calcium ([Ca(2+)]i) plays an essential role in neuronal development and function, we measured stimulus-evoked changes in [Ca(2+)]i at these differentiation durations using the Ca(2+)-responsive dye Fura-2. Increases in [Ca(2+)]i (averages ranging from 65 to 226 nM) were evoked by depolarization, ATP, l-glutamic acid, acetylcholine, and dopamine (up to 87%, 57%, 93%, 28%, and 37% responding cells, respectively) and to a lesser extent by serotonin and gamma-aminobutyric acid (both up to 10% responding cells). Notably, the changes in percentage of responsive cells and their response amplitudes over time indicate changes in the expression and functionality of the respective neurotransmitter receptors and related calcium signaling pathways during in vitro differentiation. The development of functional intercellular signaling pathways was confirmed using multielectrode arrays, demonstrating that mNPCs develop electrical activity within 1-2 weeks of differentiation (55% active wells at 14 days of differentiation; mean spike rate of 1.16 spikes/s/electrode). The combined data demonstrate that mNPCs develop functional neuronal characteristics in vitro, making it a promising model to study chemical-induced effects on the development of neuronal function.

Don't judge a neuron only by its cover: neuronal function in in vitro developmental neurotoxicity testing.

Classical cases of developmental neurotoxicity (DNT) in humans and advances in risk assessment methods did not prevent the emergence of new chemicals with (suspected) DNT potential. Exposure to these chemicals may be related to the increased worldwide incidence of learning and neurodevelopmental disorders in children. DNT is often investigated in a traditional manner (in vivo using large numbers of experimental animals), whereas development of in vitro methods for DNT reduces animal use and increases insight into cellular and molecular mechanisms of DNT. Several essential neurodevelopmental processes, including proliferation, migration, differentiation, formation of axons and dendrites, synaptogenesis, and apoptosis, are already being evaluated in vitro using biochemical and morphological endpoints. Yet, investigation of chemical-induced effects on the development of functional neuronal networks, including network formation, inter- and intracellular signaling and neuronal network function, is underrepresented in DNT testing. This view therefore focuses on in vitro models and innovative experimental approaches for functional DNT testing, ranging from optical and electrophysiological measurements of intra- and intercellular signaling in neural stem/progenitor cells to measurements of network activity in neuronal networks using multielectrode arrays. The development of functional DNT assays will strongly support the decision-making process for measures to prevent potential chemical-induced adverse effects on neurodevelopment and cognition in humans. We therefore argue that for risk assessment, biochemical and morphological approaches should be complemented with investigations of neuronal (network) functionality.