Dietborne Toxicity of Tebuconazole to a Benthic Crustacean, Heterocypris incongruens and Its Relative Contribution to the Overall Effects under Food-Water Equilibrium Partitioning.

Tebuconazole (TEB), a widely used and persistent pesticide, has garnered attention due to its frequent detection in sediments worldwide. This widespread occurrence has raised concerns about potential dietborne toxicity to benthic crustaceans, as they may ingest contaminated particles in their habitat. While bioaccumulation studies indicate the importance of TEB ingestion for benthic crustaceans, limited data exist on direct dietborne toxicity testing. This study investigated the diet-related toxicity of TEB by subjecting a benthic ostracod, Heterocypris incongruens, to a 6 day toxicity test under dietary and combined exposures. Subsequently, the importance of dietary exposure for TEB toxicity was uncovered, followed by quantification of relative dietborne toxicity contributions using a modified concentration-additive model. Results revealed that the dietary route was more toxicologically significant than the aqueous route in equilibrium. The dietborne lethal concentration (LC50) for TEB on H. incongruens was 200 (170-250) mg/kg, with an 80% relative dietborne toxicity contribution. To gain comprehensive insights into dietborne significance, toxicity data were collected from previous studies involving different pollutants to calculate relative contributions. Finally, the correlation between dietborne toxicity and the partitioning coefficient was analyzed to understand the pollutant behavior and its toxic impact when ingested through the diet.

Determining the Relative Importance of Dietborne and Waterborne Toxicity of 4-tert-Butylphenol and 4-tert-Octylphenol to the Benthic Crustacean, Heterocypris incongruens.

Organic pollutants with high solid-water equilibrium partition coefficients are adsorbed into solid particles and are easily ingested by benthic organisms, potentially causing dietborne toxicity. Whether dietborne toxicity is more important than waterborne toxicity for such chemicals remains to be determined. In this study, we identify the most relevant uptake route for the toxicity of two alkylphenols, 4-tert-butylphenol (4tBP), and 4-tert-octylphenol (4tOP). To achieve this, 6-day toxicity tests under two exposure conditions, namely dietary exposure (clean water + contaminated food) and combined exposure (contaminated water + contaminated food) were conducted on a benthic ostracod, Heterocypris incongruens. The toxicologically important exposure routes were confirmed by the consistency of dietary and aqueous dose-response relationships under different exposure conditions. During the test, frequent renewal of water and food was performed to reduce variability in the exposure conditions. The results showed that, under the equilibrium condition, the dietary exposure route was toxicologically more important than the aqueous route for 4tBP, whereas the waterborne exposure route was more important than the dietary exposure route for 4tOP. This study provides a novel approach to identify the most relevant uptake pathways for chemical toxicity, which better explains the importance of exposure routes in toxicity effects.

A Microcosm Study of Surface Sediment Environmental DNA: Decay Observation, Abundance Estimation, and Fragment Length Comparison.

Interpretation of environmental DNA (eDNA) is a major problem hindering the application of this emerging technology for environmental monitoring. The decay characteristics and bioabundance estimation of different DNA fragment lengths are largely unknown, especially for eDNA captured from surface sediments. An estuarine amphipod, Grandidierella japonica, was used as the target species in this study. We conducted a lab-scale experiment using DNA extraction to clarify the effect of bacteria on eDNA decay. We also conducted a microcosm experiment using amphipods to clarify interpretations of information regarding eDNA decay and bioabundance estimation by using two fragments with different lengths (126 and 358 bp). It was found that the bacteria concentration accelerated eDNA decay, and long fragments were more susceptible to bacteria, thus decaying faster, than short fragments in the exponential decay period. The fresh eDNA (collected within 24 h of removing the amphipods) was more indicative of bioabundance than old eDNA (collected 240-480 h after removing amphipods), and short fragments better reflected bioabundance than long fragments. Finally, we compared the half-life of eDNA in surface sediment with that in a water sample and found that the temporal scales of surface sediment and water are similar (days to weeks). Our results suggest that surface sediment also has the potential to monitor the environment at a temporal scale similar to water.

Carcinogenic profile, toxicity and source apportionment of polycyclic aromatic hydrocarbons accumulated from urban road dust in Tokyo, Japan.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in urban environments. Urban road dust (URD) generated by traffic is an important PAH accumulator. Twelve priority PAHs in < 2000 µm fraction of ten URD samples from Tokyo, Japan were characterized based on profile distributions, carcinogenicity, toxicity, and source apportionment by cluster analysis, biplot and diagnostic ratios. PAH concentrations (mg/kg dry weight) in arterial roads, highways, highway parking, highway drainage pit and residential area URD samples were 2.06-4.24, 0.25-3.37, 3.44, 4.94, and 5.26 respectively, dominated by the ∑4 rings (average 46%) and ∑5 + 6 rings (average 41%) PAHs. Biplot analysis revealed that the antecedent dry weather period, vehicle frequency and organic matter content were the dominant environmental factors governing PAH profiles of different road types. The total amount of carcinogenic PAHs in the residential URD (2.12 mg/kg) was higher than those in the arterial road (0.60-2.00 mg/kg) and highway (0.10-1.84 mg/kg) URD. Toxic equivalent concentrations (TECs) of residential, arterial road and highway URD were 0.54, (0.12-0.57), and (0.02-0.51) mg/kg, respectively. The dominant PAH sources were found to be petrogenic combustion in arterial road and highway URD, and pyrogenic combustion consisting of a mix of biomass, petroleum and traffic-related sources in the residential and highway drainage pit samples. This is also the first study to find that TEC-based toxicity should not be taken as a measure of URD toxicity.

Application of cDNA-AFLP to biomarker exploration in a non-model species Grandidierella japonica.

Biomarkers of exposure can be used to identify specific contaminants that are adversely affecting aquatic organisms. However, it remains prohibitively costly to investigate multiple novel biomarkers of exposure in a non-model species, despite the development of next-generation sequencing technology. In this study, we focused on the use of cDNA-amplified fragment length polymorphism (AFLP) as a cost-effective biomarker discovery tool to test whether it could identify biomarkers of exposure in the non-model amphipod species Grandidierella japonica. Loci were identified that were differentially expressed in amphipods exposed to reference chemicals (Cu, Zn, and nicotine) and to an environmental sample (road dust) at sublethal concentrations. Eight loci were shown to respond consistently to nicotine at different concentrations, but not to Cu or Zn. Some of the loci also responded to an environmental road dust sample containing nicotine. These findings suggest that loci identified using cDNA-AFLP could be used as biomarkers of nicotine exposure in environmental samples with complex matrices. Further studies with other organisms and toxicants are needed, but we have demonstrated that the use of cDNA-AFLP to identify biomarkers for ecotoxicological studies of non-model species is at least feasible.

Development of a chronic sediment toxicity test using the benthic ostracod Heterocypris incongruens and their application to toxicity assessments of urban road dust.

This study reports on the development and application of a chronic sediment toxicity test using the benthic ostracod Heterocypris incongruens. H. incongruens reproduction parameters such as egg production, first day of brooding, egg-laying ratio, and hatching ratio were examined in this study. The test was first validated by determining the repeatability of the test method under seven control performances. The results showed good test repeatability of most endpoints, with coefficient of variation (CV) results below 15%. However, lifetime egg production, hatching ratio, and the reproductive rate were highly variable, with CVs ranging from 29.5% to 51.9%. Next, an application example of the proposed chronic method was performed using a series of urban road dust (URD) samples diluted with a reference sediment and compared to a 6 d H. incongruens toxicity test. The results of the proposed chronic test showed a statistically significant difference in the first day of brooding at 6.25% URD which did not exhibit significant mortality and growth inhibition in 6 d toxicity test.

Effect of salinity on the toxicity of road dust in an estuarine amphipod Grandidierella japonica.

Urban runoff can reach coastal aquatic environments; however, little is known about the effect of salinity on road runoff toxicity. The objective of this study is to investigate the toxicity of highway road dust over a salinity gradient from 5 to 35‰, in an estuarine benthic amphipod, Grandidierella japonica. Road dust toxicity was evaluated by assessing mortality after 10 days of exposure and short-term microbead ingestion activity of the amphipod. For all road dust samples considered, amphipod mortality increased with increasing salinity, whereas no significant difference in mortality was observed among test salinities in the reference river sediment. Ingestion activity during exposure to road dust decreased with increasing salinity. In fact, none of the individuals ingested any microbeads at salinity of 35‰. If assumed microbead ingestion is a proxy for feeding activity, high mortality at 35‰ could be attributed to aquatic exposure and not to dietary exposure. These findings suggest that road dust may have considerable impact on benthic organisms at high salinity levels.

Variation in PAH patterns in road runoff.

Twelve particle-bound polycyclic aromatic hydrocarbons (PAHs) were measured in the first flush regime of road runoff during nine events in Winterthur in Switzerland. The total PAH contents ranged from 17 to 62 µg/g. The PAH patterns measured at different time intervals during the first flush periods were very similar within each event irrespective of variation in suspended solids (SS) concentration within the first flush regime. However, the PAH patterns were different from event to event. This indicates that the environment plays an important role in PAH accumulation in SS. A toxicity identification evaluation approach using a toxicity equivalency factor (TEF) was applied to compare toxicities in the different events. The TEFs were found to be between 8 and 33 µg TEQ g(-1) (TEQ: toxic equivalent concentration). In some cases, two events having similar total PAH contents showed two fold toxicity differences.

Selection of formulated sediment and feeding condition for 10-day spiked-sediment toxicity test with estuarine amphipod Grandidierella japonica.

This study was performed to select formulated sediment with appropriate feeding conditions for the evaluation of estuarine sediment toxicity. Grandidierella japonica, which inhabits seawater worldwide, was examined for 10-day survival, and the repeatability was evaluated under nine test conditions. The formulated sediment feeding condition combined with the highest survival rate was applied to evaluate fluoranthene toxicity in the sediment. The KB-T10 feeding condition showed the highest survival rate of 93.5%, with sufficient repeatability (4.6%). In the fluoranthene spiked-sediment toxicity test, the estimated LC50 (95% significance level) was successfully achieved under the KB-T10 condition but not under the OE-T10 condition. Therefore, we propose the use of TetraMin® (1 mg/org./day) and Kemble sediment in 10-day static spiked-sediment toxicity tests with G. japonica. Further improvements are needed for OECD sediment in seawater to increase the survival rate and prevent chemical loss due to overlying water renewal.

Pathogens and disease vectors/hosts monitoring in aquatic environments: Potential of using eDNA/eRNA based approach.

Infectious diseases are spreading in to previously unreported geographical regions, and are reappeared in regions 75 or 100 years after their last reported case, as a result of environmental changes caused by anthropogenic activities. A pathogen, vector/host monitoring methodology is therefore indispensable in identifying potential transmission sites, providing early warnings and evaluating the human health risks of these infectious diseases in a given area. Recently, environmental DNA (eDNA) and environmental RNA approach (eRNA) have become widespread in monitoring organisms in the environment due to advantages like lower cost, time, and labour requirements. However, eDNA/eRNA based monitoring of pathogens and vectors/hosts using aquatic samples is limited to very few studies. In this review, we summarized the currently available eDNA/eRNA based human and non-human pathogens and vectors/hosts detection studies in aquatic samples. Species-specific shedding, transport, and decay of eDNA/eRNA in aquatic environments which is essential in estimating the abundance of pathogen, vectors/host in focus is also summarized. We also suggest the usage of eDNA/eRNA approach in urban aquatic samples like runoff in identifying the disease vectors/hosts inhabiting in locations which are not accessible easily.

Development and application of a metabolomic tool to assess exposure of an estuarine amphipod to pollutants in the environment.

Identifying major adverse effects on aquatic organisms in environmental samples is still challenging, and metabolomic approaches have been utilized as non-target screening techniques in the context of ecotoxicology. While existing methods have focused on statistical tests or univariate analysis, there is the need to further explore a multivariate analytical method that captures synergetic effects and associations among metabolites and toxicants. Here we show a new tool for screening sediment toxicity in the environment. First, we constructed predictive models using the metabolomic profiles and the result of exposure tests, to discriminate the toxic effects of target substances. The developed models were then applied to sediment samples collected from an actual urban area that contain chromium, nickel, copper, zinc, cadmium, fluoranthene, nicotine, and osmotic stress, incorporated with exposure tests of the benthic amphipod Grandidierella japonica. As a result, the fitted models showed high predictive power (Q2 > 0.71) and could detect toxicants from mixed chemical samples across a wide range of concentrations in test datasets. The application of the constructed models to river sediment and road dust samples indicated that almost all target substances were less toxic compared with the effects at LC50 levels. Only zinc showed slight increasing trends among samples, suggesting that the proposed method can be used for prioritization of toxicants. The present work made a direct connection between chemical exposures and metabolomic responses, and draws attention to the need for further studies on interactive mechanisms of metabolites in toxicological assessments.

Comparison of metal (Zn and Cu) complexation characteristics of DOM in urban runoff, domestic wastewater and secondary effluent.

This study was aimed at comparing the Zn and Cu binding parameters with dissolved organic matter (DOM) in road runoff, wastewater treatment plant (WWTP) effluent and influent. Conditional stability constant (K') and binding site concentration ([L]) are important to predict free or labile metal concentration and toxicity in the water environment. The values of K' and [L] of three DOMs were determined by metal titration and Scatchard linearization. The Zn and Cu titration data for DOMs in WWTP effluent and influent fitted to a two-ligand model, while DOM in road runoff fitted to a single-ligand model. The order of the values of K' was WWTP influent > WWTP effluent > road runoff both for Zn and Cu. Total ambient binding site concentration ([L(T)]) was lower in DOM of road runoff (1.4-1.5 µM) than those in DOM of WWTP effluent (1.6-2.3 µM) and influent (17-18 µM), suggesting lower metal complexation capacity in DOM of road runoff. DOM in WWTP influent was expected to bind to both Zn and Cu more strongly than that of the effluent DOM.

Simultaneous screening for chemically diverse micropollutants in public water bodies in Japan by high-performance liquid chromatography-Orbitrap mass spectrometry.

An improved assessment of environmental risks to public water bodies requires screening a large number of micropollutants. This study reports the development of a novel target screening method based on solid-phase extraction (SPE), HPLC, and high-resolution Orbitrap MS for the analysis of micropollutants with diverse chemical properties. First, target compounds were screened for their detectability by Orbitrap MS. An optimized SPE cartridge and HPLC column maximized recovery and separated most target compounds. The sensitivity and repeatability of the method was validated by determining the detection limits and relative standard deviation (RSD). Eighty-four compounds with highly diverse properties were simultaneously detected with detection limits of 0.1-100 ng/L. Of these compounds, 52 were quantitated, with R2 ≥ 0.99 by linearity analysis and SPE recovery ratios of ≥50%. The remaining 32 compounds were qualitatively detected, with R2 < 0.99 or SPE recovery ratio of <50%. Satisfactory repeatability was obtained (RSD < 13.5%). This method was applied to the surveillance of the Arakawa River in Japan in 2019. Thirty-two compounds, including pesticides, surfactants, plasticizers, adhesives, and industrial solvents, were detected in the river. The measured concentrations of 13 compounds were compared with their predicted no effect concentrations (PNECs). Decanoic acid showed a higher concentration than the corresponding PNEC value, suggesting that its risk to the Arakawa water environment required further evaluation. The concentrations of dicyclohexylamine, 1,3-diphenylguanidine, and 2,4-dichlorophenoxyacetic acid were higher than their corresponding PNEC/10 values, demonstrating that these compounds were of higher priority than other compounds.

Effect of calcite saturation state on the growth and mortality of Heterocypris incongruens and a proposal for an reference artificial sediment in the sediment toxicity test ISO14371.

Since the establishment of the whole-contact sediment toxicity test using Heterocypris incongruens in 2012, this user-friendly method has been widely used for the assessment of many kinds of solid samples, such as sediment, soil, compost, and sewage sludge. There are two endpoints for this method: mortality and growth inhibition. Currently, there is no standard toxicity threshold established for the endpoint of growth inhibition. However, there is evidence showing that the calcite saturation state of the overlying water, which might be different among various samples, influences the growth and survival of H. incongruens. Thus, it is necessary to characterize the influence of the calcite saturation state to ensure that the test results are reliable and comparable among different samples. In the present study, we created artificial sediments comprised of quartz sand and 0-20% calcite particulates to manipulate the calcite saturation state in the overlying water and performed the test using H. incongruens for 6 d. The results show that a low calcite saturation state inhibited the growth of H. incongruens but had no effect on survival rate. The growth of ostracods was linearly related to the calcite saturation index of the overlying water. Additionally, a formula for artificial sediment consisting of 10% calcite and 90% quartz sand for the H. incongruens toxicity test was proposed. This study helps to distinguish H. incongruens growth inhibition caused by calcite saturation from that caused by toxicity.

Whole transcriptome analysis of an estuarine amphipod exposed to highway road dust.

Urban road dust can potentially have adverse effects on aquatic and benthic ecosystems if discharged into receiving waters; however, little is known about the mode of action of road dust toxicity within aquatic organisms. With an aim to reveal the biological effects of road dust on benthic crustacean species, we performed a de novo transcriptome analysis of the estuarine amphipod Grandidierella japonica exposed to road dust collected from highways around Tokyo. A transcriptome analysis by Illumina HiSeq 2500 identified differentially expressed genes related to the gamma-aminobutyric acid (GABA) signaling pathway, oxidative damage, and cuticle metabolism. Among these, a GABAB receptor subunit showed down-regulation in the road dust treatment, but a constant expression in the treatment of road dust with a carbonaceous resin XAD-4, which can reduce the acute toxicity of road dust to G. japonica. These results and the time course expressions of the related genes were partially confirmed by quantitative PCR (qPCR) experiments. Although the linkage between acute lethal toxicity and the molecular initiating events induced by road dust was still unclear, our findings provide lines of evidence to identify the causative toxicants in urban road dust.

Impact of holding time on toxicity change of urban road dust during runoff process.

During runoff process, the urban road dust (URD) passes through the road-side gutters and detention tanks, where it gets hold for a certain period of time, hours to weeks, before making its way into the water bodies. A part of the water-exchangeable toxicants are leached by the water, and some strongly bound toxicants remain attached to the URD. Toxicity of urban runoff is dependent on holding time, water volume, and the type and composition of the wet road dust (WeRD) and leachate. However, there are no studies that have elucidated the manner in which toxicities of the WeRD and leachate vary during prolonged holding in the runoff processes. The main objectives of this research were to, i) identify the distribution of toxicants in the WeRD and leachate, and ii) evaluate the change in toxicity during prolonged holding. The URD samples that were collected from residential road, arterial road and highways in Tokyo, Japan, were mixed with moderately hard water in varying ratios (1:2 to 1:16) and were held for a certain time (1h to 5days) before centrifuging into the WeRD and leachate. Toxicity test was conducted with ostracod Heterocypris incongruens direct contact test. Toxicity of both the WeRD and leachate from residential area was not significantly greater than the 20% lethal limit. Toxicity of the WeRD from other stations initially decreased, further reached a minimum corresponding to the critical holding time, and it subsequently increased again. Toxicity of the arterial leachate gradually increased, whereas that of the highway leachate gradually decreased during 5days holding. Unionized ammonia and zinc were confirmed as one of the possible toxicants. This study proposes and verifies a model for the toxicity change of the WeRD during prolonged holding.

Performance of a membrane biofilm reactor for denitrification with methane.

In this study, a membrane biofilm reactor was investigated for aerobic methane oxidation coupled indirectly to denitrification, a process potentially useful for denitrification of nitrate-contaminated waters and wastewaters using methane as external electron donor. Methane and oxygen were supplied from the interior of a silicone tube to a biofilm growing on its surface. We found that the membrane biofilm reactor was to some extent self-regulating in the supply of methane and oxygen. Although the intramembrane partial pressures of methane and oxygen were varied, the oxygen-to-methane ratio penetrating the membrane tended towards 1.68. Both nitrate removal rate and dissolved organic carbon (DOC) production rate appeared to be positively correlated with intramembrane methane pressure. Based on measured nitrate removal rates, DOC production rates, and nitrate removal efficiency, the possibility of using this method for treatment of a hypothetical wastewater was evaluated.

The sorption of heavy metal species by sediments in soakaways receiving urban road runoff.

Infiltration facilities are designed for both the retention of non-point pollutants and the replenishment of groundwater in urban areas. In this study, sorption tests were conducted to evaluate the speciation of heavy metals and their behaviour in infiltration facilities receiving urban road runoff containing high DOC concentrations and stable heavy metal organic complexes. Road dust and three soakaway sediments were collected from heavy traffic areas and a residential area with an infiltration-type sewage system in Tokyo, Japan. Sequential multiple batch tests were conducted by adding prepared road dust leachate (artificial road runoff) or deionised water to soakaway sediment to obtain soakaway sediment leachate (artificial percolating water from soakaway sediment), which mimicked the sorption by sediments in soakaways receiving urban road runoff. Heavy metal speciation was assessed by means of a combination of anion-exchange resin measurements and MINTEQA2 model calculations, and further validated by chelating resin measurements. In road dust leachates and soakaway sediment leachates, Cu predominantly existed as organic complexes and carbonates, whereas most Mn, Zn and Cd were found to exist in the form of free ions and carbonate complexes. Stable organic complexes of Cu in road dust leachates were strongly adsorbed by soakaway sediments despite the limited adsorption of DOC. On the other hand, desorption of free Mn, Zn and Cd ions from the sediment receiving road dust leachates was observed, indicating that heavy metals such as Mn, Zn and Cd may ultimately reach groundwater as free ions.

Diversity of catechol 2,3-dioxygenase genes of bacteria responding to dissolved organic matter derived from different sources in a eutrophic lake.

Catechol 2,3-dioxygenase (C23O) is an extradiol dioxygenase that plays an important role in degrading aromatic compounds such as those found at polluted sites. However, little is known about the diversity of C23O genes in unpolluted environments. In such environments, various factors, including the quality and quantity of dissolved organic matter (DOM), could influence the composition and behaviour of bacterial community possessing C230 genes. We investigated C23O genes in bacteria responding to DOM from various sources in a eutrophic lake by PCR and cloning. Six microcosms filled with lake water containing indigenous bacteria and DOM from different sources were incubated for 10 days. After 1 or 2 days of incubation, C23O genes were detected in the microcosms enriched with DOM recovered from inflow river water and humus from reed grass. The sequences were very diverse but had features conserved in extradiol dioxygenases. The clone libraries generated on day 2 showed distinctive compositions among microcosms, indicating that bacteria possessing a variety of C23O genes responded differently to DOM from different sources. After 10 days of incubation, C23O genes in a previously unidentified gene cluster, 'Cluster X', became dominant in the libraries.

Identification of particles containing chromium and lead in road dust and soakaway sediment by electron probe microanalyser.

Individual particles containing Cr and/or Pb and other major components were identified in road dust from a heavily used road (hereinafter 'heavy traffic road dust'), road dust from a residential area and soakaway sediment by electron probe microanalyser to locate their sources and carrier particles. Individual particles containing high levels of Cr and/or Pb (>or=0.2%) were identified using wavelength dispersive spectrometry (WDS) map analysis. Chromium, Pb and other major elements were then determined by means of a combination of WDS and energy-dispersive spectrometry in all identified particles, 50 particles containing neither Cr nor Pb from each type of road dust and soakaway sediment, and yellow road line markings. WDS map analysis revealed that many particles containing both Cr and Pb were present among the identified particles in heavy traffic road dust, whereas they were minor components in road dust from the residential area and soakaway sediment. The plots of X-ray intensities of Cr vs. Pb were linear for the identified particles containing both Cr and Pb in heavy traffic road dust, and the line closely fitted the plots for the three yellow road line marking samples. Individual particles were then classified using cluster analysis of element components. The results revealed that the adsorption of source materials or released metals onto soil minerals occurred in road dust and soakaway sediment, that the yellow road line markings were sources of Cr and Pb in heavy traffic road dust, and that materials containing Fe as a major component, such as stainless steel, were additional sources of Cr in both road dust and soakaway sediment.