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.

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.

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.

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.

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.

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.

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.

Duration of life-cycle toxicity tests with the ostracod Heterocypris incongruens.

An acute sediment toxicity test using the ostracod Heterocypris incongruens is user friendly and has high sensitivity; however, a life-cycle test using this species has not been developed. The most challenging problem when developing a life-cycle test is that the egg development time varies greatly and is sometimes too long (> 150 d) to monitor. It is desirable to shorten the duration of life-cycle toxicity tests including the observation period of egg development while preserving the ecological relevance of the net reproductive rate (R0 ), an endpoint in the life-cycle test. Therefore, in the present study, we suggest a practical test duration for R0 using population growth rate (λ) as a measure of ecological relevance. We collected a range of life history characteristics of the ostracod by combining data from the literature and experimental results, constructed population matrix models, and calculated λ for 20 life history patterns. The results showed that a longer test period (> 150 d) did not increase the correlation coefficients between R0 and λ. Rather, a shorter test duration resulted in R0 being highly correlated with λ. Our results suggest that a life-cycle toxicity test using the ostracod can provide an ecologically relevant toxicity endpoint, even if the test is abandoned after approximately 50 d and unhatched eggs remain. Environ Toxicol Chem 2017;36:3443-3449. © 2017 SETAC.

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.

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.

Causes of highway road dust toxicity to an estuarine amphipod: Evaluating the effects of nicotine.

Urban road dust can potentially have adverse effects on ecosystems if it is discharged into receiving waters. This study investigated the causes of highway road dust toxicity by performing sediment toxicity identification evaluation (TIE) tests with an estuarine amphipod, Grandidierella japonica. In addition to metals and polycyclic aromatic hydrocarbons, which are traditionally considered to be the major toxicants in road runoff, we focused on dissolved nicotine as a causative toxicant. The sediment TIE results suggested that organic contaminants contributed to the majority of toxicity, and that the contribution of unionized nicotine to the toxicity was the highest among the chemicals considered. However, additional mortality tests with 48-h pulsed nicotine exposure demonstrated that exposure to nicotine at the same concentration as the baseline level in TIE tests did not cause significant 10-day amphipod mortality. Thus, the road dust toxicity could not be explained only by unionized nicotine, thereby suggesting contributions from joint effects of the measured toxicants and the presence of other unmeasured factors.

Blue carbon in human-dominated estuarine and shallow coastal systems.

Estuarine and shallow coastal systems (ESCS) are recognized as not only significant organic carbon reservoirs but also emitters of CO2 to the atmosphere through air-sea CO2 gas exchange, thus posing a dilemma on ESCS's role in climate change mitigation measures. However, some studies have shown that coastal waters take up atmospheric CO2 (Catm), although the magnitude and determinants remain unclear. We argue that the phenomenon of net uptake of Catm by ESCS is not unusual under a given set of terrestrial inputs and geophysical conditions. We assessed the key properties of systems that show the net Catm uptake and found that they are often characteristic of human-dominated systems: (1) input of high terrestrial nutrients, (2) input of treated wastewater in which labile carbon is highly removed, and (3) presence of hypoxia. We propose that human-dominated ESCS are worthy of investigation as a contributor to climate change mitigation.

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.

Characterization of toxicants in urban road dust by Toxicity Identification Evaluation using ostracod Heterocypris incongruens direct contact test.

The current study involves characterization of organic compounds, heavy metals, and ammonia as potential toxicants in one arterial road (St. 3) and two highway (St. 7 and 8) urban road dust (URD) collected in Tokyo, Japan. URD toxicity was evaluated by Toxicity Identification Evaluation using the ostracod Heterocypris incongruens direct contact test. URDs were treated with resins (10% XAD-4, 20% SIR-300 and 20% SIR-600) to determine the reduction in mortality after treatment. The mortality of ostracods exposed to St. 3 URD (baseline 80%) was significantly (p<0.05) reduced to 0% and 47% after XAD-4 and SIR-300 treatment respectively. This reduction led us to suspect hydrophobic organic compounds and heavy metals as potential toxicants. Subsequent elution of the recovered XAD-4 with polar (methanol, acetone) and non-polar (dichloromethane) solvents confirmed the dominance of relatively polar hydrophobic organic toxicants. The dissolved concentration of Cu and Zn after SIR-300 treatment exceeded the 50% lethal concentration (LC50) for ostracods, which led us to suspect Cu and Zn as other probable toxicants. The concentration of Zn in a SIR-300 acidic elutriate, recovered after the termination of toxicity test, confirmed Zn as one of the toxicants in St. 3. The baseline mortality (100%) of St. 7 was significantly reduced (23%) by SIR-300 treatment. This indicated the presence of heavy metals as the probable toxicant. However, the concentration of dissolved heavy metals (Cd, Cr, Cu, Ni, Pb and Zn) in the overlying water was below previously reported LC50 values for each metal in St. 7. Recovery of Zn concentrations exceeding the LC50 in the St. 7 SIR-300 elutriate led us to suspect a dietary exposure route of Zn to the ostracod during the direct contact test. The overall results indicate that the toxicant types can vary widely depending on the road sampled.

Seawater-driven forward osmosis for enriching nitrogen and phosphorous in treated municipal wastewater: effect of membrane properties and feed solution chemistry.

Seawater-driven forward osmosis (FO) is considered to be a novel strategy to concentrate nutrients in treated municipal wastewater for further recovery as well as simultaneous discharge of highly purified wastewater into the sea with low cost. As a preliminary test, the performance of FO membranes in concentrating nutrients was investigated by both batch experiments and model simulation approaches. With synthetic seawater as the draw solution, the dissolved organic carbon, phosphate, and ammonia in the effluent from a membrane bioreactor (MBR) treating municipal wastewater were 2.3-fold, 2.3-fold, and 2.1-fold, respectively, concentrated by the FO process with approximately 57% of water reduction. Most of the dissolved components, including trace metals in the MBR effluent, were highly retained (>80%) in the feed side, indicating high water quality of permeate to be discharged. The effect of membrane properties on the nutrient enrichment performance was investigated by comparing three types of FO membranes. Interestingly, a polyamide membrane possessing a high negative charge demonstrated a poor capability of retaining ammonia, which was hypothesized because of an ion exchange-like mechanism across the membrane prompted by the high ionic concentration of the draw solution. A feed solution pH of 7 was demonstrated to be an optimum condition for improving the overall retention of nutrients, especially for ammonia because of the pH-dependent speciation of ammonia/ammonium forms. The modeling results showed that higher than 10-fold concentrations of ammonia and phosphate are achievable by seawater-driven FO with a draw solution to feed solution volume ratio of 2:1. The enriched municipal wastewater contains nitrogen and phosphorous concentrations comparable with typical animal wastewater and anaerobic digestion effluent, which are used for direct nutrient recovery.

Comparison of aquatic and dietary exposure of heavy metals Cd, Cu, and Zn to benthic ostracod Heterocypris incongruens.

The benthic ostracod Heterocypris incongruens is becoming an important tool for the ecotoxicological assessment of contaminated sediments. However, no study has yet explored solid-phase (dietary) exposure to ostracod. The present study examined the effects of metals on H. incongruens through aquatic and dietary exposures. The algal food Chlorella vulgaris was exposed to different concentrations of cadmium (Cd), copper (Cu), and zinc (Zn), and subcellular distributions of these metals in algal cells were determined. Parallel experiments were also performed to determine the toxic effects of dissolved metals on ostracod through aqueous exposure. Significant toxic effects on mortality were observed when ostracod were fed with Cd-contaminated (47-100%) and Cu-contaminated algae (55-100%). With increasing accumulated Zn in the algal cell, ostracod mortality also increased (20-83%). Aquatic exposure of ostracod to different concentrations of dissolved Cd (3.2-339 µg Cd/L) and Cu (260-2600 µg Cu/L) resulted in high observed mortalities (57-100% and 95-100%, respectively). Based on the results of aqueous and dietary exposure tests, it can be concluded that both exposure routes are important in evaluating the toxic effect of Cd on ostracod. Similar results were observed in tests using other metals (i.e., using Cu and Zn), thus emphasizing the importance of considering not only aquatic but also dietary exposure routes when evaluating metal toxicity to ostracod.