Understanding the toxicity risk of antibiotic emissions of aquaculture from the perspective of fluctuations concentration.

Organisms are generally exposed to target contaminant with stable concentrations in traditional ecotoxicological studies. However, it is difficult to truly represent the dynamics and complexity of actual aquatic pollution for risk management. Contaminants may enter nearby aquatic systems in pulsed exposure, thus resulting in that aquatic organisms will be exposed to contaminants at fluctuating concentrations. Especially during the season of summer, due to the changes in displacement or periodic emissions of veterinary antibiotics in aquaculture, algal blooms occur frequently in surrounding waters, thus leading to eutrophication of the water. Florfenicol (FFC) is currently widely used as a veterinary antibiotic, but the aquatic ecological risks of FFC under concentration fluctuations are still unknown. Therefore, the acute exposure, chronic exposure and pulsed exposure effects of FFC on Microcystis aeruginosa were investigated to comprehensively evaluate the ecological risk of FFC and raise awareness of the pulsed exposure mode. Results indicated that the toxic effects of FFC on M. aeruginosa were dominated by exposure mode, exposure duration, exposure frequency, and exposure concentration. The maximum growth inhibition rate of the 10 µg/L FFC treatment amounted to 4.07% during chronic exposure of 18 days. However, the growth inhibition rate decreased from 55.1% to 19.31% when algae was exposure to 10 µg/L FFC during the first pulsed exposure (8 h). Therefore, when the concentration of FFC was equal under chronic and pulsed exposure, FFC exhibited greater toxicity on M. aeruginosa in short pulsed exposure than in continuous exposure. In addition, repetitive pulsed exposure strengthened the resistance of M. aeruginosa on FFC. The adaptive regulation of algae was related to the duration and frequency of exposure. Above results suggested that traditional toxicity assessments lacked consideration for fluctuating concentrations during pollutant emissions, thus underestimating the environmental risk of contaminant. This investigation aims to facilitate the standardization of pulsed exposure.

Toxicity of Wildland Fire-Fighting Chemicals in Pulsed Exposures to Rainbow Trout and Fathead Minnows.

Intrusions of fire-fighting chemicals in streams can result from containment and suppression of wildfires and may be harmful to native biota. We investigated the toxicity of seven current-use fire-fighting chemicals to juvenile rainbow trout (Oncorhynchus mykiss) and fathead minnows (Pimephales promelas) by simulating chemical intrusions under variable field conditions to provide insights into the potential damage these chemicals may cause in waterways. In three separate attenuated exsposure assays in which chemical concentration decreased throughout the 96-h exposure period, we varied water flow rate, water hardness, and initial concentration of test chemical. In an additional series of four pulsed exposure assays in which fish encounter chemical for up to 1 h followed by an observation period in control water, we altered concentration of test chemical, water temperature, duration of chemical exposure, and number of exposures to determine delayed toxicity or recovery. Mortality of rainbow trout was higher across treatments at a warmer temperature and also increased with increasing concentration rate, increasing exposure duration, and sequential exposures across assays. For fathead minnows, mortality increased with increasing concentration of fire retardant and longer exposure durations. Because the ratio of toxic un-ionized ammonia to ionized ammonia is greater with increasing temperature and pH, future studies could investigate the effects of water temperature and pH on native fishes under environmentally relevant concentrations of fire-fighting chemicals. Environ Toxicol Chem 2022;41:1711-1720. Published 2022. This article is a U.S. Government work and is in the public domain in the USA.

Effects of continuous and intermittent cadmium exposure on HPGL axis, GH/IGF axis and circadian rhythm signaling and their consequences on reproduction in female zebrafish: Biomarkers independent of exposure regimes.

Typical biomarkers of cadmium (Cd) pollution have well been confirmed in fish from continuous exposure pattern. However, in a natural environment, fish may be exposed to Cd intermittently. In this study, juvenile female zebrafish were exposed for 48 days to 10 µg/L Cd continuously, 20 µg/L for 1 day in every 2 days or 30 µg/L for 1 day in every 3 days. The toxic effects were evaluated using 8 various physiological and biochemical endpoints like specific growth rate (SGR), 17ß-estradiol (E2) and vitellogenin (VTG) concentrations in plasma, reproductive parameters (gonadosomatic index (GSI), egg-laying amount, spawning percentage, and hatching and mortality rate of embryos). Transcription of 59 genes related to hypothalamic-pituitary-gonadal-liver (HPGL) axis, circadian rhythm signaling and insulin-like growth factor (IGF) system was examined. SGR, spawning percentage, E2 and VTG levels declined in fish exposed to 10 and 20 µg/L Cd but remained relatively stable in fish exposed to 30 µg/L Cd. Exposure to 10, 20 and 30 µg/L Cd significantly reduced GSI, hatching rate and mortality rate. Similarly, mRNA expression of 27 genes were sensitive to both continuous and intermittent Cd exposure. Among these genes, expression levels of 10 genes had more than 5-fold increase or decrease, including mRNA levels of vtg1, vtg2, vtg3, esr1, igf2a, igf2b, igfbp5b, nr1d1, gnrh3 and gnrhr4. The most sensitive molecular biomarker was vtg3 expression with 1500-3100 fold increase in the liver. The present study, for the first time, provides effective candidate biomarkers for Cd, which are independent of exposure regimes.

Pulsed exposure toxicity testing: Baseline evaluations and considerations using copper and zinc with two marine species.

Methods to assess environmental impacts from episodic discharges on receiving water bodies need a more environmentally relevant and scientifically defensible toxicity test design. Many permittees are regularly required to conduct 96-h toxicity tests on discharges associated with events that are generally less than 24 h in duration. Current standardized methods do not adequately reflect these episodic discharge conditions at either the point of compliance nor as it mixes with the receiving environment. In order to evaluate more representative biological effects, an alternative toxicity approach is described incorporating pulsed exposures of effluents and subsequent transfer of test organisms to clean water for the remainder of the test. This pulsed exposure protocol incorporates a slight modification to USEPA Whole Effluent Toxicity (WET) chronic and acute methods for two marine species, purple sea urchin embryos, Strongylocentrotus purpuratus, and juvenile mysid shrimp Americamysis bahia. Tests were performed with toxicants using standard static (96 h) and pulsed (6, 12, and 26 h) exposures. Following pulsed exposures, organisms were transferred to uncontaminated seawater for the remainder of the 96-h test period. Results for these species and endpoints indicated that the sensitivity of these species to copper and zinc were up to two orders of magnitude greater using standard continuous exposures compared to shorter pulsed exposures. Additional considerations assessed included timing of the onset of a pulse and latent effects following an exposure. This modified approach requires minimal modification to current standard methods and increases the realism to more accurately assess toxic effects resulting from episodic discharges.

The effect of salinity fluctuation in freshwater streams on the fecundity of post-diapause Chironomus dilutus.

Much scientific research dedicated to understanding the effects of freshwater salinization caused by road de-icing salts has utilized static exposures, with many tests conducted at winter or spring temperatures. While relevant for lentic ecosystems, pulsed patterns of salinity occur in lotic environments, particularly in summer months where precipitation can decrease elevated salinity levels caused by retention of residual salts. The current study aimed to evaluate the effects of pulsed patterns of salinity on the emergence, sex ratio, and fecundity of Chironomus dilutus over two generations of laboratory exposure. Three road de-icing salt treatments, including a control, modeled after environmental monitoring data of two local streams, were used to determine the ecological effects of periodic declines in salinity on C. dilutus at summer temperatures. No significant effects were observed on emergence success or sex ratios within or across generations, but fecundity of C. dilutus in the high salt treatment was reduced regardless of generation (P < 2e-16), possibly due to increased osmoregulatory stress caused by increased salinities. The intermediate and decreasing salinities may account for the lack of negative effects on emergence success and sex ratios by protecting sensitive life stages. More research is needed on long-term effects of reduced fecundity on population viability. The current study suggests more research using a similar experimental design is needed to fully evaluate the influence of road de-icing salts in lotic environments, as static laboratory exposures may not accurately reflect environmental changes in salinity.

Developmental exposure window influences silver toxicity but does not affect the susceptibility to subsequent exposures in zebrafish embryos.

Silver is a non-essential, toxic metal widespread in freshwaters and capable of causing adverse effects to wildlife. Its toxic effects have been studied in detail but less is known about how sensitivity varies during development and whether pre-exposures affect tolerance upon re-exposure. We address these knowledge gaps using the zebrafish embryo (Danio rerio) model to investigate whether exposures encompassing stages of development prior to mid-blastula transition, when chorion hardening and epigenetic reprogramming occur, result in greater toxicity compared to those initiated after this period. We conducted exposures to silver initiated at 0.5 h post fertilisation (hpf) and 4 hpf to determine if toxicity differed. In parallel, we exposed embryos to the methylation inhibitor 5-azacytidine as a positive control. Toxicity increased when exposures started from 0.5 hpf compared to 4 hpf and LC50 were significantly lower by 1.2 and 7.6 times for silver and 5-azacyitidine, respectively. We then investigated whether pre-exposure to silver during early development (from 0.5 or 4 hpf) affected the outcome of subsequent exposures during the larvae stage, and found no alterations in toxicity compared to naïve larvae. Together, these data demonstrate that during early development zebrafish embryos are more sensitive to silver when experiments are initiated at the one-cell stage, but that pre-exposures do not influence the outcome of subsequent exposures, suggesting that no long-lasting memory capable of influencing future susceptibility was maintained under our experimental conditions. The finding that toxicity is greater for exposures initiated at the one-cell stage has implications for designing testing systems to assess chemical toxicity.

Time-Variable Exposure Experiments in Conjunction with Higher Tier Population and Effect Modeling to Assess the Risk of Chlorotoluron to Green Algae.

An algae population model was applied to describe measured effects of pulsed exposure to chlorotoluron on populations of Pseudokirchneriella subcapitata in 2 laboratory flow-through chemostat tests with different exposure regimes. Both tests enabled evaluation of adverse effects on algae during the exposure and population recovery afterward. Impacts on population densities after chlorotoluron exposure were directly visible as biomass loss in the chemostats. Recovery was observed after each exposure peak. The test results indicate that P. subcapitata is unlikely to show an increased sensitivity to chlorotoluron after pulsed exposure. No altered response or adaptation of the algae to chlorotoluron was observed, with the exception of the last high peak in flow-through test 2. Therefore, an adaptation to the test substance cannot be excluded after long-term exposure. However, recovery to the steady-state level after this peak indicates that the growth rate (fitness) was not significantly reduced in the population with higher tolerance. No differences in chlorotoluron impact on the populations over time in terms of growth were detected. Model predictions agreed well with the measured data. The tests and modeling results validate the model to simulate population dynamics of P. subcapitata after pulsed exposure to chlorotoluron. Model predictions and extrapolations with different exposure patterns are considered reliable for chlorotoluron. The good reproducibility of the population behavior in the test systems supports this conclusion. An example modeled extrapolation of the experimental results to other (untested) exposure scenarios shows a potential approach to using the validated model as a supportive tool in risk assessment. Environ Toxicol Chem 2019;38:2520-2534. © 2019 SETAC.

Predicting the survival of zebrafish larvae exposed to fluctuating pulses of lead and cadmium.

Aquatic organisms are often exposed to time-varied concentrations of contaminants due to pulsed inputs in natural water. Traditional toxicology experiments are usually carried out in a constant exposure pattern, which is inconsistent with the actual environment. In this study, a refined toxicokinetic-toxicodynamic (TK-TD) model was used to study the toxic effects of Pb and Cd on zebrafish larvae under three pulse exposures with 2, 4, and 6 h, respectively. The parameter sensitivity analysis showed that JM, max had the greatest impact on the output of the model. Cd or Pb pulse exposure resulted in less death than constant exposure at the same time-weighted average (TWA) concentrations. Survival fraction in larvae under 6 h interval between two pulses of Pb or Cd was larger than that under 2 h and 4 h interval. Toxicity under constant exposure of Cd or Pb was greater than that under 2, 4, and 6 h interval pulse exposure because the cumulative Cd or Pb concentration in the body under constant exposure was greater than that under pulse exposure. The results also showed that the stochastic death (SD) model was more suitable than the individual tolerance (IT) model for predicting the survival fraction of larvae under pulse exposure to Pb and Cd, which was indicated by higher R2 (0.670-0.940) in SD model than that (0.588-0.861) in IT model. Our model provides approaches for laboratory toxicity testing and modeling approaches for addressing the toxicity of heavy metal pulsed exposure.

Comparison of the effects of constant and pulsed exposure with equivalent time-weighted average concentrations of the juvenile hormone analog pyriproxyfen on the reproduction of Daphnia magna.

Short-term pulsed exposure tests have been increasingly used to evaluate the ecotoxicity of pollutants of which concentrations vary over time in the field. In pulsed exposure, time-weighted average (TWA) concentration is often used as an index of exposure. However, there have been few studies to demonstrate whether TWA concentration can be used to evaluate the effect of endocrine-disrupting chemicals on the daphnids. Pyriproxyfen is one of the juvenile hormone analogs that induces daphnids to produce male offspring. To evaluate whether peak or TWA concentration can explain the effects of pyriproxyfen on daphnid reproduction, we measured the number of offspring and the proportion of male offspring produced by Daphnia magna during 21-day under different exposure treatments, constant, single-pulse, and multi-pulse exposure, at an equivalent TWA concentration. Constant exposure of 50 ng/L pyriproxyfen did not affect either the fecundity or the proportion of male offspring, while a single-pulse exposure of 525 ng/L pyriproxyfen over 2 day at four different age did not reduce fecundity, but the proportion of male offspring increased age dependently. Multi-pulses exposure of 131 ng/L pyriproxyfen over two days four times (total eight days) resulted in a decrease in fecundity and the highest proportion of male offspring. Daily observation demonstrated that male offspring was only produced several days after the exposure to a certain concentration of pyriproxyfen. Therefore, neither TWA nor peak concentration accurately evaluated the effects of pulsed exposure of pyriproxyfen on the reproduction of D. magna, particularly its effect on the proportion of male offspring.

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.

Potential risks to freshwater aquatic organisms following a silvicultural application of herbicides in Oregon's Coast Range.

Glyphosate, aminomethylphosphonic acid (AMPA), imazapyr, sulfometuron methyl (SMM), and metsulfuron methyl (MSM) were measured in streamwater collected during and after a routine application of herbicides to a forestry site in Oregon's Coast Range. Samples were collected at 3 stations: HIGH at the fish-no-fish interface in the middle of the harvest and spray unit, MID at the bottom of the unit, and LOW downstream of the unit. All herbicides were applied by helicopter in a single tank mix. AMPA, imazapyr, SMM, and MSM were not detected (ND) in any sample at 15, 600, 500, and 1000 ng/L, respectively. A pulse of glyphosate peaking at approximately equal to 62 ng/L manifested at HIGH during the application. Glyphosate pulses peaking at 115 ng/L (MID) and 42 ng/L (HIGH) were found during the first 2 postapplication storm events 8 and 10 days after treatment (DAT), respectively: glyphosate was less than 20 ng/L (ND) at all stations during all subsequent storm events. All glyphosate pulses were short-lived (4-12 h). Glyphosate in baseflow was approximately equal to 25 ng/L at all stations 3 DAT and was still approximately equal to 25 ng/L at HIGH, but ND at the other stations, 8 DAT: subsequently, glyphosate was ND in baseflow at all stations. Aquatic organisms were subjected to multiple short-duration, low-concentration glyphosate pulses corresponding to a cumulative time-weighted average (TWA) exposure of 6634 ng/L × h. Comparisons to TWA exposures associated with a range of toxicological endpoints for sensitive aquatic organisms suggests a margin of safety exceeding 100 at the experimental site, with the only potential exception resulting from the ability of fish to detect glyphosate via olfaction. For imazapyr, SMM, and MSM the NDs were at concentrations low enough to rule out effects on all organisms other than aquatic plants, and the low concentration and (assumed) pulsed nature of any exposure should mitigate this potential. Integr Environ Assess Manag 2017;13:396-409. © 2016 SETAC.

Comparisons of discrete and integrative sampling accuracy in estimating pulsed aquatic exposures.

Most current-use pesticides have short half-lives in the water column and thus the most relevant exposure scenarios for many aquatic organisms are pulsed exposures. Quantifying exposure using discrete water samples may not be accurate as few studies are able to sample frequently enough to accurately determine time-weighted average (TWA) concentrations of short aquatic exposures. Integrative sampling methods that continuously sample freely dissolved contaminants over time intervals (such as integrative passive samplers) have been demonstrated to be a promising measurement technique. We conducted several modeling scenarios to test the assumption that integrative methods may require many less samples for accurate estimation of peak 96-h TWA concentrations. We compared the accuracies of discrete point samples and integrative samples while varying sampling frequencies and a range of contaminant water half-lives (t50 = 0.5, 2, and 8 d). Differences the predictive accuracy of discrete point samples and integrative samples were greatest at low sampling frequencies. For example, when the half-life was 0.5 d, discrete point samples required 7 sampling events to ensure median values > 50% and no sampling events reporting highly inaccurate results (defined as < 10% of the true 96-h TWA). Across all water half-lives investigated, integrative sampling only required two samples to prevent highly inaccurate results and measurements resulting in median values > 50% of the true concentration. Regardless, the need for integrative sampling diminished as water half-life increased. For an 8-d water half-life, two discrete samples produced accurate estimates and median values greater than those obtained for two integrative samples. Overall, integrative methods are the more accurate method for monitoring contaminants with short water half-lives due to reduced frequency of extreme values, especially with uncertainties around the timing of pulsed events. However, the acceptability of discrete sampling methods for providing accurate concentration measurements increases with increasing aquatic half-lives.

Calibration of nylon organic chemical integrative samplers and sentinel samplers for quantitative measurement of pulsed aquatic exposures.

Environmental exposures often occur through short, pulsed events; therefore, the ability to accurately measure these toxicologically-relevant concentrations is important. Three different integrative passive sampler configurations were evaluated under different flow and pulsed exposure conditions for the measurement of current-use pesticides (n=19), polyaromatic hydrocarbons (n=10), and personal care products (n=5) spanning a broad range of hydrophobicities (log Kow 1.5-7.6). Two modified POCIS-style samplers were investigated using macroporous nylon mesh membranes (35µm pores) and two different sorbent materials (i.e. Oasis HLB and Dowex Optipore L-493). A recently developed design, the Sentinel Sampler (ABS Materials), utilizing Osorb media enclosed within stainless steel mesh (145µm pores), was also investigated. Relatively high sampling rates (Rs) were achieved for all sampler configurations during the short eight-day exposure (4300-27mL/d). Under flow conditions, median Rs were approximately 5-10 times higher for POCIS-style samplers and 27 times higher for Sentinel Samplers, as compared to static conditions. The ability of samplers to rapidly measure hydrophobic contaminants may be a trade off with increased flow dependence. Analyte accumulation was integrative under pulsed and continuous exposures for POCIS-style samplers with mean difference between treatments of 11% and 33%; however, accumulation into Sentinel Samplers was more variable. Collectively, results show that reducing membrane limitations allows for rapid, integrative accumulation of a broad range of analytes even under pulsed exposures. As such, these sampler designs may be suitable for monitoring environmental substances that have short aquatic half-lives.

The induction of metallothioneins during pulsed cadmium exposure to Daphnia magna: Recovery and trans-generational effect.

Although the importance of pulse exposure has gained ground in recent years, there were few studies on recovery and trans-generational effect of it. Two successive generations Daphnia magna were exposed to cadmium (Cd) pulses for 6h at the concentrations from 40 to 100 µg/l. The changes of tolerance and induction of MTs in exposed D. magna and their offspring were measured. The reduced tolerance of exposed D. magna was returned to levels similar to control after about 9 days in a generation. The level of MT still increased up to 3 days after exposure. In the experimental range, exposure duration played a decisive role in MT induction. The tolerance of F1 was lower than F0 and decreased with increasing pulsed concentrations of F0. Exposed to the same pulse, the MT levels of F1 were higher than the MT levels of F0, but the more obvious detoxification of MT in F1 had not been found. Our results suggest that pulsed cadmium exposure had impact on offspring of exposed organism and the risk assessment should take trans-generational effect into account.

Sensitivity of a green alga to atrazine is not enhanced by previous acute exposure.

Exposure to atrazine in small lotic systems can be episodic, with short-term pulses (peaks) followed by lower, decreasing concentrations. Algae and macrophytes recover rapidly from pulsed exposure to atrazine, but reported observations of population response to subsequent exposures are minimal and inconclusive. Consequently, the sensitivity of Pseudokirchneriella subcapitata to atrazine following a pulsed exposure was assessed. Exposure concentrations reflected amplifications of those observed in streams from highly vulnerable watersheds in regions of intense use. Initial pulsed atrazine exposure at 0, 150 or 300 µg/L for 24-h was followed by 72-h exposure to 0, 5, 10, 25, or 50 µg/L. Measured responses were cell density, growth rate, chlorophyll-a, and maximum quantum yield of photosystem II. Algal recovery was rapid and prior pulsed exposure to atrazine did not significantly affect subsequent sensitivity (EC10s, EC25s) for any endpoint, indicating no changes in tolerance at the population level for this species.

Highly time-variable exposure to chemicals--toward an assessment strategy.

Organisms in the environment experience fluctuating, pulsed, or intermittent exposure to pollutants. Accounting for effects of such exposures is an important challenge for environmental risk assessment, particularly given the simplified design of standard ecotoxicity tests. Dynamic simulation using toxicokinetic-toxicodynamic (TK-TD) models describes the processes that link exposure with effects in an organism and provides a basis for extrapolation to a range of exposure scenarios. In so doing, TK-TD modeling makes the risk assessment more robust and aids use and interpretation of experimental data. Toxicokinetic-toxicodynamic models are well-developed for predicting survival of individual organisms and are increasingly applied to sublethal endpoints. In the latter case particularly, linkage to individual-based models (IBMs) allows extrapolation to population level as well as accounting for differences in effects of toxicant exposure at different stages in the life cycle. Extrapolation between species remains an important constraint because there is currently no systematic understanding of species traits that cause differences in the relevant processes. Toxicokinetic-toxicodynamic models allow interrogation of exposure profiles to determine intrinsic toxicity potential rather than using absolute maximum concentrations or time-weighted averages as surrogates. A decision scheme is proposed to guide selection of risk assessment approaches using dose extrapolation based on Haber's Law, TK-TD models, and/or IBMs depending on the nature of toxic effect and timing in relation to life history.