Sublethal Endpoints Research Articles

Embryotoxic and proteotoxic effects of water and sediment from the Neckar River (Southern Germany) to zebrafish (Danio rerio) embryos

The Neckar River (Southern Germany) represents an aquatic system strongly affected by numerous anthropogenic activities. Thus, it is an excellent model for ecotoxicological investigations. The present study aims to assess time and spatial variations of embryo- and proteotoxic effects in surface water and sediment. For this end, embryos of zebrafish (Danio rerio) were exposed to Neckar River samples collected in the Tubingen region in different seasons over 2 years. Additionally, quantification of the heat shock (stress) protein Hsp70 was carried out in newly hatched larvae; furthermore, physico-chemical water parameters were measured in order to gain baseline information about the limnologic conditions. Nearly all of the investigated Neckar River sites caused elevated mortality, developmental retardation and failures, modified heart rate and reduced hatching success in zebrafish embryos and larvae. Additionally, exposure to Neckar River water and sediment led to changes in larval Hsp70 level. During the 2 years of investigation, seasonal differences of embryo- and proteotoxic effects occurred. Along these lines, physico-chemical measurements delivered basic information for the interpretation of in vivo test data. Our study suggests a changing toxic burden in the whole investigated study area. Consequently, for ecotoxicological field studies, time and spatial variations on small scale must be dealt with. The lethal and sublethal endpoints of the fish embryo test combined with Hsp70 level measurements proved to be effective tools for toxicity assessment of environmental samples.

Assessment of Jatropha curcas L. biodiesel seed cake toxicity using the zebrafish (Danio rerio) embryo toxicity (ZFET) test

Consequent to the growing demand for alternative sources of energy, the seeds from Jatropha curcas remain to be the favorite for biodiesel production. However, a significant volume of the residual organic mass (seed cake) is produced during the extraction process, which raises concerns on safe waste disposal. In the present study, we assessed the toxicity of J. curcas seed cake using the zebrafish (Danio rerio) embryotoxicity test. Within 1-h post-fertilization (hpf), the fertilized eggs were exposed to five mass concentrations of J. curcas seed cake and were followed through 24, 48, and 72hpf. Toxicity was evaluated based on lethal endpoints induced on zebrafish embryos namely egg coagulation, non-formation of somites, and non-detachment of tail. The lowest concentration tested, 1g/L, was not able to elicit toxicity on embryos whereas 100% mortality (based also on lethal endpoints) was recorded at the highest concentration at 2.15g/L. The computed LC50 for the J. curcas seed cake was 1.61g/L. No further increase in mortality was observed in the succeeding time points (48 and 72hpf) indicating that J. curcas seed cake exerted acute toxicity on zebrafish embryos. Sublethal endpoints (yolk sac and pericardial edema) were noted at 72hpf in zebrafish embryos exposed to higher concentrations. The observed lethal endpoints induced on zebrafish embryos were discussed in relation to the active principles, notably, phorbol esters that have remained in the seed cake even after extraction.

Suspended sediment effects on walleye (Sander vitreus)

Environmental windows are seasonal restrictions imposed on dredging operations in the Great Lakes and other waterways of the United States. Such restrictions often generically assume that sediments resuspended by dredging result in adverse impacts to fish; this is the case in western Lake Erie where the environmental window was established due to potential impacts on walleye (Sander vitreus). To address this concern, laboratory studies mimicking sediment resuspension during dredging operations in western Lake Erie were conducted to determine whether suspended sediments affect walleye eggs and fingerlings. Newly laid eggs and 45- to 60-d old fingerlings from separate hatcheries were exposed for 72-h under flow-through conditions to 0, 100, 250, and 500mg/L total suspended sediment (TSS). Fingerlings, eggs, and newly hatched larvae were analyzed for multiple lethal and sublethal endpoints. Data indicated no significant effects of suspended sediment on egg hatch success or fingerling survival after three days of exposure. No significant differences were observed when comparing percent egg viability in the control with any TSS treatment; however, a downward trend was observed at 500mg/L. No significant differences were observed during comparisons of fingerling gill lamellae in the control with any TSS treatment; however, a statistically significant difference was observed when comparing gill lamellae in the control with the original supply animals. No significant differences were found between control means and unexposed eggs or any TSS treatment. These effect data for walleye will serve to better inform the setting of environmental windows for this species in western Lake Erie.

Biomonitoring toxicity of natural sediments using juvenile Hyalella curvispina (Amphipoda) as test species: evaluation of early effect endpoints

The utility of early effect endpoints as biomarkers of ecotoxicity of natural sediments in water-sediment static system was investigated. The particular goal was to evaluate the ecotoxicity of the sediment samples from La Choza stream, located in upper basin of the Reconquista river, the second most polluted river of Argentina. Native juveniles Hyalella curvispina were used as test organisms evaluating survival, growth, oxidative stress parameters (SOD; CAT, TBARS) and the electron transport system (ETS) activity as early toxic effect. This study used methodologies and techniques that allow the assessment of sediment pollution with a native species as test organism and provided data to discuss the viability of sublethal endpoints as tools for freshwater sediment assessment. In spring and in summer two ten-day series of whole-sediment assays were conducted simultaneously: (a) standard assays and (b) biomarkers assays. A control sediment was ran simultaneously in which no--effect on survival was measured. In summer there was a significant increase in length and biomass in both exposed and control groups. In spring an inhibitory effect on growth and an increase in oxidative damage with a concomitant rise in antioxidant defenses, was observed in animals exposed to La Choza sediment. ETS measurement indicated a significant depression of metabolic activity of amphipods exposed to contaminated sediments. The measured biomarkers represent the first record for juvenile H. curvispina exposed to polluted natural sediments under standardized laboratory conditions. The used bioanalytical tools demonstrated higher sensitivity and a more accurate assessment of the effects than those obtained by the standard tests of survival and growth. We propose their adoption in biomonitoring of freshwater sediment toxicity.

Investigating lethal and sublethal effects of the trace metals cadmium, cobalt, lead, nickel and zinc on the anemone Aiptasia pulchella, a cnidarian representative for ecotoxicology in tropical marine environments

The zooxanthellate sea anemone Aiptasia pulchella is found throughout the tropical and subtropical oceans of the Indo-Pacific and is easily maintained in aquaria, posing potential suitability as a standard tropical marine test organism for use in ecotoxicology. To gain an understanding of the sensitivity of A. pulchella to trace metals, 96-h static-renewal toxicity tests were conducted. Values of 96-h LC50 between 946 and 1196 µg L–1 were estimated for cadmium, between 595 and 1146 µg L–1 for zinc, 8060 and 12 352 µg L–1 for lead and 2209 and 5751 µg L–1 for nickel. In addition, preliminary assessment of rapid tentacle retraction was made. Six-hour EC50 values of 355 and 979 µg L–1 for cadmium, between 384 and 493 µg L–1 for zinc, between 2340 and 2584 µg L–1 for nickel, and 2610 µg L–1 for lead, were estimated for ‘severe’ tentacle retraction. Cobalt concentrations up to 1547 µg L–1 caused extreme zooxanthellae loss, but no more than 10% mortality and no rapid ‘severe’ tentacle retraction. The present study has provided important baseline information, enabling comparison of the acute sensitivity of A. pulchella to trace metals with other marine invertebrates, and guiding the development of sublethal endpoints.

Joint toxicity of permethrin and cypermethrin at sublethal concentrations to the embryo-larval zebrafish

Pyrethroids, the widely used pesticides, are highly toxic to aquatic organisms. However, little information is so far available regarding the joint toxicity of type I and type II pyrethroids to fish. Zebrafish is a well-accepted aquatic vertebrate model for toxicity assessment due to small size, easy husbandry, high fecundity and transparent embryos. In this study, we utilized embryo-larval zebrafish to elucidate the combined effects of sublethal concentrations of permethrin (PM) and cypermethrin (CP), which are the most frequently used type I and type II pyrethroids, respectively. Fish were exposed from 3h postfertilization (hpf) to 144 hpf to binary mixtures of nominal concentrations of 100, 200, 300μgL−1 PM (PM100, PM200, PM300) and 10, 20, 30μgL−1 CP (CP10, CP20, CP30). Analytical data of the real concentrations of the chemicals showed a significant degradation of the pyrethroids but an obvious recovery after the renewal of the exposure solution. Defect rates of embryos exposed to these low concentrations of single PM or CP exhibited no statistically significant difference from the control,while the application of combination of PM and CP resulted in deleterious effects on zebrafish embryonic development. In all PM200 and PM300 exposure groups, increasing CP concentrations acted additively to the action of PM in terms of all sublethal endpoints. Co-treatment of embryos with the specific sodium channel blocker MS-222 and pyrethroids (individuals or the mixture) caused a decline in the incidences of body axis curvature and spasms compared to treatment of animals with pyrethroids alone, suggesting that the developmental toxicity of PM and CP to zebrafish was related to disruption of ion channels. We further revealed that mixture of the two pyrethroids caused greater down-regulation in the mRNA levels of proneural genes. The individual pesticides had no effect on the activity of superoxide dismutase (SOD), while the mixture exposure caused significant induction. Treatment with CP or the mixture increased the activity of catalase (CAT). Taken together, our data indicated that the mixture of PM and CP caused higher incidence of morphological defects, greater inhibition in proneural gene expression and more oxidative stress, compared to the single chemical at the corresponding doses. Our findings suggest that the combination of type I and type II pyrethroids poses a greater risk to fish in the water column.

Application of a Unique Test Design to Determine the Chronic Toxicity of Boron to the Aquatic Worm Lumbriculus variegatus and Fatmucket Mussel Lampsilis siliquoidea

The chronic (21- and 28-day) toxicity of boron was determined for two freshwater benthic macroinvertebrates: the fatmucket mussel Lampsilis siliquoidea and the aquatic worm Lumbriculus variegatus. The rapid depletion of boric acid from spiked sediments in tests using flow-through overlying waters was addressed by constant addition of boric acid to overlying water at concentrations matching those of the targeted porewater exposures. This proved highly successful in maintaining constant whole-sediment and sediment porewater boron concentrations. Boron sublethal 25% inhibition concentration values based on porewater concentrations were 25.9mg B/L (L. variegatus) and 38.5mg B/L (L. siliquoidea), indicating similar test organism sensitivity. Expressed as dry whole-sediment values, the respective L. variegatus and L. siliquoidea sublethal (growth) IC25 values for whole-sediment exposures were 235.5mg B/kg sediment dry weight (dw) and 310.6mg B/kg dw. The worm lethality-based end points indicated greater sensitivity than the sublethal end points, bringing into question the validity of a "lethality" end point for L. variegatus given its fragmentation mode of reproduction. For comparison, water-only mussel exposures were tested resulting in an IC25 value of 34.6mg B/L, which was within 20% of the porewater value. This suggests that the primary route of boron exposure was through the aqueous phase. The results of this study indicated that for test materials that are readily water soluble, standard sediment test designs may be unsuitable, but water-only exposures can provide toxicological data representative of sediment tests.

Behavioural, physiological and biochemical markers in damselfly larvae (Ischnura elegans) to assess effects of accumulated metal mixtures

Currently it is not known at which organismal level effects of metal mixtures in nature can best be detected, which is relevant to develop accurate monitoring schemes and quality standards. The present study investigated relationships between accumulated metals with different levels of biological organisation in the aquatic larval stage of the damselfly Ischnura elegans. Larvae were collected in seven Flemish ponds differing in metal load. In each field-collected larva we quantified concentrations of accumulated metals and a set of biochemical markers (acetylcholinesterase (AChE) and glutathione-S-transferase (GST)), physiological endpoints (energy storage), and behavioural responses (locomotory activity and the feeding rate). Accumulated metal levels and the measured endpoints significantly differed among ponds, however, a large variation in metal load index was observed within individuals of the same population. Only GST and energy availability could be partly predicted by the observed variation in metal load index on individual damselfly level. However, no single endpoint could be used to detect the observed variation in metal load index among populations. In conclusion, the sublethal endpoints cannot be used as reliable biomarkers to monitor the toxicity of accumulated metal mixtures in natural populations of I. elegans.

Exposure–dose–response of Tellina deltoidalis to metal contaminated estuarine sediments 2. Lead spiked sediments

Lead accumulation in estuarine sediments, as a result of activities such as mining and ore smelting, and through urban runoff is a continuing problem in the increasingly developed world. Marine organisms accumulate lead, which is known to be highly toxic to biological processes and to degrade organism and ecosystem health. Here the relationship between lead exposure, dose and response was investigated in the sediment dwelling, deposit feeding, marine bivalve Tellina deltoidalis. Bivalves were exposed in the laboratory to individual lead spiked sediments at < 0.01, 100 and 300μg/g dry mass, for 28days and accumulated total tissue lead concentrations of 4, 96 and 430μg/g, respectively. Subcellular fractionation indicated that around 70% of the total accumulated tissue lead was detoxified, three quarters of the detoxified lead fraction was converted into metal rich granules, with the remainder in the metallothionein like protein fraction. The majority of biologically active lead was associated with the mitochondrial fraction with up to a 128 fold increase in lead burden in exposed organisms compared to controls. This indicates lead detoxification was occurring but the organism was unable to prevent lead interacting with sensitive organelles. With increased lead exposure T. deltoidalis showed a suppression in glutathione peroxidase activity, total glutathione concentration and reduced GSH:GSSG ratios, however, these differences were not significant. Lead exposed T. deltoidalis had a significantly reduced total antioxidant capacity which corresponded with increased lipid peroxidation, lysosomal destabilisation and micronuclei frequency. The exposure–dose–response relationships demonstrated for lead exposed T. deltoidalis supports its potential for the development of sublethal endpoints in lead toxicity assessment.

Toxicity and critical body residues of Cd, Cu and Cr in the aquatic oligochaete Tubifex tubifex (Müller) based on lethal and sublethal effects

The aim of the present study was to estimate critical body residues (CBRs) of three metals [cadmium (Cd), copper (Cu), chromium (Cr)] in the aquatic oligochaete Tubifex tubifex based on lethal (LBR) and sublethal effects (CBR), and to discuss the relevance of the exposure to sediment for deriving CBR. Toxicity parameters (LC50, EC50, LBR50 and CBR50) were estimated for each metal by means of data on survival and on several sublethal variables measured in short-term (4 days), water-only exposures and in long-term, chronic (14 and 28 days) exposures using metal-spiked sediment. Sublethal endpoints included autotomy in short-term exposure, as well as reproduction and growth in chronic bioassays. LBR50 and CBR50 were 3-6 times higher in sediment than in water-only exposure to Cd and about 2-11 times higher for Cu, depending on the measured endpoint; however, for Cr these parameters varied only by a factor of 1.2. Cu and Cr LBR50 and CBR50 values in 96 h water-only exposure were very similar (survival 2.39 μmol Cu g(-1) dw, 2.73 μmol Cr g(-1) dw; autotomy 0.53 μmol Cu g(-1) dw, 0.78 μmol Cr g(-1) dw). However, in metal-spiked sediments, 28 d CBR50 values for autotomy, reproduction and growth ranged 6.76-29.54 μmol g(-1) dw for Cd, 3.88-6.23 μmol g(-1) dw for Cu, 0.65 μmol g(-1) dw for Cr (calculated only on total number of young). Exposure conditions (time and presence/absence of sediment) seem to be influential in deriving metal CBR values of Cd and Cu, while appear to be irrelevant for Cr. Thus, CBR approach for metals is complex and tissue residue-toxicity relationship is not directly applicable so far.

Is Atyaephyra desmarestii a useful candidate for lethal and sub-lethal toxicity tests on pharmaceutical compounds?

Single and mixture toxicity tests on three pharmaceutical compounds, Diclofenac (DF), Ibuprofen (IB) and Carbamazepine (CBZ), were carried out with the freshwater shrimp Atyaephyra desmarestii. Lethal and sublethal responses were analyzed for single compounds. Lethal concentrations (LC50) obtained for each individual compound, after 96h of exposure, were 6.3mgL−1 for DF, 13.3mgL−1 for IB and 94.3mgL−1 for CBZ. The selected sublethal endpoints of food ingestion, osmoregulatory capacity and respiration rates were not affected by the exposures to environmentally-relevant concentrations. Based on mortality data obtained, the predictive no effect concentration (PNEC) was calculated for each of the compounds, and compared with predicted environmental concentrations (PEC) reported in surface waters. The environmental risk of each compound was estimated as the ratio between PEC/PNEC, and indicated that IB could represent a medium risk in freshwater environments. Additionally, binary and ternary mixture toxicity assays of the selected compounds were carried out. The data obtained was applied to two predictive toxicity models: Concentration Addition (CA) and Independent Action (IA). Finally, risk assessment was estimated using risk quotients (RQ) for the compound mixtures based on EC50 and LC50 values.

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.

Influence of water temperature and net tending frequency on the condition of fish bycatch in a small-scale inland commercial fyke net fishery

To date, most studies of commercial fisheries bycatch have focused on mortality at time of capture as an endpoint. However, sub-lethal indicators of organismal condition have the potential to reveal mechanisms associated with mortality (both at time of capture and post-release) and opportunities for improving fish welfare. In this study, we simulated commercial fishing efforts in inland lakes with fyke nets during a typical fishing season (early April to late June) in southeastern Ontario, Canada, where bycatch of non-target fish species had previously been documented. Using non-target gamefish (i.e., largemouth bass [Micropterus salmoides, Lacépède], northern pike [Esox Lucius, L.]), as well as a target species (i.e., bluegill [Lepomis macrochirus, Rafinesque]), we examined the sub-lethal consequences of capture (e.g., blood physiology, reflex impairment, and injury) and compared the effects of being retained in the net for two different durations (i.e., two or six days) over a range of water temperatures (i.e., 3–28°C). Sub-lethal physiological disturbances (i.e., blood glucose and lactate) in largemouth bass and bluegill tended to be greater at higher water temperatures. However, fish retained for six days generally did not exhibit greater stress than those retained for two days, with the exception of plasma glucose in largemouth bass. Reflex impairment was similar among temperature and retention periods. Fish retained in nets experienced a range of injuries (including fin frays, scale loss, and mouth damage) that had the potential to facilitate the development of opportunistic pathogenic infections. Greater incidences of injury on fish bycatch tended to be associated with higher temperatures and longer retention. To reduce physiological disturbances and injury that could lead to delayed mortality, we suggest that regulations for inland commercial fishers require them to check their nets more frequently as water temperatures increase. We suggest that future studies of bycatch incorporate sub-lethal endpoints given that they serve as an objective measure of fish welfare and can provide quantitative mechanistic information to support management actions.

Occurrence of copper acclimation in the least killifish Heterandria formosa, and associated biochemical and physiological mechanisms

We investigated the occurrence of copper acclimation in the least killifish, Heterandria formosa using both lethal and sublethal endpoints. We also investigated potential mechanisms underlying the observed acclimation. To assess the occurrence of acclimation, fish were exposed to either a background Cu level or to 15μg/L Cu for seven days and subsequently exposed to a lethal Cu level (150μg/L Cu). During the latter exposure, fish were monitored for survival till all fish had died, and (during the first 8h of this exposure) for changes in whole-body Na levels and lipid peroxidation (LPO). During the high-level Cu exposure, fish pre-exposed to copper had a significantly longer time-to-death than did the control fish. Similarly, neither whole-body Na nor LPO changed in the Cu-pre-exposed fish during the 8h of the exposure to 150μg/L Cu – while both decreased significantly in the control fish. Thus, acclimation was evident for both time-to-death and the sublethal endpoints. These results also indicate that Cu toxicity may involve both Na loss and LPO, and that Cu-acclimation may be brought about by prevention of these effects. Our follow-up study on potential mechanisms underlying this copper acclimation used a similar pre-exposure/exposure design. Fish were subsampled at the end of the 7-day acclimation period – just before the commencement of high-level Cu exposure (T0), after 4h of this Cu exposure (T4), and again after 8h of this Cu exposure (T8). Whole-body Cu accumulation, Na+/K+-ATPase activity, metallothionein levels, and catalase activity were quantified for these time points. While Cu levels were higher in the Cu-pre-exposed fish than in the control fish at T0, net Cu accumulation was faster in the control fish than in the Cu-pre-exposed fish during the subsequent high-level Cu exposure. Consequently, changes in Cu accumulation dynamics may play a role in the resistance. Metallothionein induction may also play a role in the observed acclimation, as Cu-acclimated fish had a significantly higher metallothionein concentration compared to the control fish. There was no evidence of involvement of Na+/K+-ATPase in the acclimation, as the activity of this enzyme remained lower in the pre-exposed fish than in the control fish throughout both Cu exposure periods. There was limited evidence that a reduced loss of catalase activity plays a role in the acclimation; catalase activity did not differ after the pre-exposure period but was significantly higher in Cu-acclimated fish than in the control fish at T8.

Toxicity of Quantum Dots and Cadmium Salt to Caenorhabditis elegans after Multigenerational Exposure

To fully understand the biological and environmental impacts of nanomaterials requires studies that address both sublethal end points and multigenerational effects. Here, we use a nematode to examine these issues as they relate to exposure to two different types of quantum dots, core (CdSe) and core-shell (CdSe/ZnS), and to compare the effect to those observed after cadmium salt exposures. The strong fluorescence of the core-shell QDs allowed for the direct visualization of the materials in the digestive track within a few hours of exposure. Multiple end points, including both developmental and locomotive, were examined at QD exposures of low (10 mg/L Cd), medium (50 mg/L Cd), and high concentrations (100 mg/L Cd). While the core-shell QDs showed no effect on fitness (lifespan, fertility, growth, and three parameters of motility behavior), the core QDs caused acute effects similar to those found for cadmium salts, suggesting that biological effects may be attributed to cadmium leaching from the more soluble QDs. Over multiple generations, we commonly found that for lower life-cycle exposures to core QDs the parents response was generally a poor predictor of the effects on progeny. At the highest concentrations, however, biological effects found for the first generation were commonly similar in magnitude to those found in future generations.

Relative potency of PCB126 to TCDD for sublethal embryotoxicity in the mummichog (Fundulus heteroclitus)

The relative potency (ReP) of 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for sublethal responses was assessed in Fundulus heteroclitus embryos. Eggs were treated with intravitelline injections of graded sublethal doses of PCB126 (312–5000pgg−1 wet weight, ww) or TCDD (5–1280pgg−1 ww). At 16 days post-fertilization (DPF), craniofacial deformities were observed in larvae hatched from eggs treated with the two highest doses of PCB126 (2500–5000pgg−1 ww). Both compounds caused a dose-responsive reduction of larval growth and prey capture ability (at ≥1250pgg−1 ww), and induction of ethoxyresorufin-O-deethylase (EROD) activity (at ≥80pgg−1 ww). The dose–response relationships for EROD activity for PCB126 and TCDD had similar slopes and the ReP of PCB126 to TCDD for EROD activity was estimated at 0.71. This is 140-fold higher than the World Health Organization (WHO) TCDD equivalency factor (TEF) of PCB126 for fish (0.005), which is based on rainbow trout (Oncorhynchus mykiss) embryolethality data. The slope of the dose–response relationship for prey capture ability for PCB126 was steeper than for TCDD, suggesting different mechanisms of action. Expression levels of several genes were also studied by quantitative real-time polymerase chain reaction (qPCR) following exposure to single doses of TCDD or PCB126 (1280 and 1250pgg−1 ww, respectively) causing similar EROD induction. A different pattern of responses was observed between PCB126 and TCDD: PCB126 appeared to induce antioxidant responses by inducing sod2 expression, while TCDD did not. These results suggest that relative potencies are species-specific and that the current ReP for PCB126 underestimates its toxicity for some fish species. It is recommended to develop species-specific RePs for a variety of sublethal endpoints and at environmentally relevant doses.

Antioxidant defense system responses and DNA damage of earthworms exposed to Perfluorooctane sulfonate (PFOS)

The use of earthworms as a sublethal endpoint has significantly contributed to the ecological risk assessment of contaminated soils. Few studies have focused on the potential toxicity of PFOS to earthworms in the soil. In this work, artificial soils were tested, and contact filter paper studies were used. The results showed that earthworm growth was generally inhibited. The antioxidant activities of the enzymes superoxide dismutase, peroxidase, catalase and glutathione peroxidase were initially activated and then inhibited. Reduced glutathione content was observed, and malondialdehyde content was elevated over the duration of the exposure. These results suggested that PFOS induced oxidative stress in earthworms. In addition, the values of olive tail moment, tail DNA% and tail length using SCGE showed similar frequency distributions and increased with increases in the PFOS concentration. These results suggest that all concentrations of PFOS cause DNA damage.

Analysis of burrowing ability as a sublethal endpoint in a marine sediment bioassay with Corophium volutator (Pallas)

Purpose Acute whole-sediment bioassay with the estuarine and marine amphipod Corophium volutator (Pallas) is widely used to assess toxicity of sediments. According to the guidelines DIN EN ISO 16712, mortality is the determined toxic endpoint. Additionally, the reburrowing ability of the surviving organisms of this acute toxicity test in fresh uncontaminated sediment is suggested as the sublethal endpoint, but insufficient information (e.g., exact measurement protocols) on this endpoint is provided, thus confounding factors and the interpretation of the results. The aim of this study was to provide information on burrowing activity as a sublethal endpoint.

Toxicity of 15 veterinary pharmaceuticals in zebrafish (Danio rerio) embryos

Extensive use of veterinary pharmaceuticals may result in contamination of water bodies adjacent to pasture land or areas where animal manure has been applied. In order to evaluate the potential risk to fish embryos 15 veterinary pharmaceuticals were investigated by use of an extended zebrafish embryo toxicity test. Chemical analysis of the exposure medium was performed by solid phase extraction–liquid chromatography–tandem mass spectrometry (SPE–LC–MS/MS) for 11 of the compounds and potential metabolism by the embryos was studied for albendazole, febantel, fenbendazole and oxfendazole. Newly fertilized zebrafish eggs were exposed under static conditions in 96-well plates for 6 days to the pharmaceuticals: 5 antibacterials and 10 antiparasitics. Endpoints including mortality, malformations and other sublethal responses were recorded at 24, 48 and 144h post fertilization (hpf). The pharmaceuticals causing the highest toxicity were antiparasitics whereas the tested antibacterials, danofloxacin, enrofloxacin, tylosine, trimethoprim and oxytetracyclin had a much lower toxic potency in zebrafish embryos. Most toxic were fenbendazole, albendazole and flumethrin with no observed effect concentrations (NOECs) around 0.02mg/L. The overall NOEC was determined by lethality for the following pharmaceuticals: albendazole, fenbendazole and oxfendazole. Sublethal endpoints, including malformations, side-laying embryos, tremors, reduced movements and altered heart rate increased the sensitivity of the tests and determined the overall NOECs for febantel, doramectin, ivermectin, flumethrin and toltrazuril. Exposure to doramectin and ivermectin caused a decrease in movements at 24hpf and a decrease in heart rate at 48hpf. Flumethrin exposure resulted in decreased time to hatching, except at the highest concentrations, and caused an increase in heart rate at 48hpf. In contrast, toltrazuril caused an increased time to hatching and a decrease in heart rate. Chemical analysis of the exposure medium after the tests revealed great differences between nominal and measured concentrations, emphasizing the need of including analysis of the actual exposure concentrations. The results indicated that metabolism of albendazole into its sulfoxide protected the embryos from toxicity. Albendazole was metabolized efficiently into albendazole sulfoxide at lower exposure concentrations, resulting in reduced toxicity. At higher concentrations, an increasing proportion of albendazole remained unmetabolized and embryo mortality occurred. Metabolism by the embryos of febantel into fenbendazole and oxfendazole and of fenbendazole into oxfendazole was demonstrated. It is suggested that the toxic effect of febantel in zebrafish embryos is due to metabolism into fenbendazole.

Aiptasia pulchella: a tropical cnidarian representative for laboratory ecotoxicological research

An urgent need exists to identify suitable tropical marine species for use in the development of sensitive and reliable test methods for routine laboratory ecotoxicological testing. Corals are a group of organisms not represented in routine ecotoxicology due to inherent difficulties in laboratory husbandry, and sea anemones from the same phylum (cnidaria) may be useful proxies. Aiptasia pulchella is a tropical symbiotic sea anemone with a wide geographic range. It is well suited to laboratory conditions and has been used extensively in research. However, its suitability as a toxicity test species has not been investigated. Assessment of juvenile recruitment in laboratory and semi-outdoor conditions showed higher production in semi-outdoor conditions; however, laboratory rearing produced enough recruits to run routine toxicity tests. In investigations of the sensitivity of A. pulchella to contaminants, acute tests were conducted on 1- to 2-mm juveniles using copper. Lethal concentration, 50% (LC50) values at 96 h estimated from tests using five and 10 replicates ranged from 30 to 83 and 60 to 90 µg/L, respectively, and a 28-d LC50 of 26 µg/L was estimated. During the present study, sublethal endpoints were investigated; chronic assessment of inhibited asexual reproduction looks promising (12-d effective concentration, 50% [EC50] 15 µg/L) and should be assessed further. Aiptasia pulchella is a species worthy of investigation as a cnidarian representative, and will be an invaluable contribution to tropical marine ecotoxicologists.