Effects of life cycle exposure to polystyrene microplastics on medaka fish (Oryzias latipes).

The number of published studies evaluating the effects of microplastics (MPs) in fish has increased in the last decade. However, of the available studies, few have explored the long-term effects of MPs on fish growth and reproduction and have resorted to MPs in the form of µm-sized beads/microspheres. In this study, 6-10 day-old post-hatch medaka (Oryzias latipes) fish were exposed to 50 (i.e. 1X) and 500 (i.e. 10X) µg of heterogeneously sized and irregularly shaped virgin polystyrene (PS) MP particles (200-µm range)/L for 150 days. These concentrations corresponded to respective daily mean values of 247 and 3087 particles/L administered through the diet. The PS MPs dietary exposure resulted in body burdens of 114 and 440 particles/g fish on day 50, and of 78 and 173 particles/g fish on day 100 since the respective exposures to the 1X and the 10X treatments started. The biometric analyses found no incidence of PS MPs ingestion on overall fish growth and development. The histological survey in the 10X group did not reveal alterations in gills or in the digestive tract. Mild alterations in other organs were seen and included increased fluid material in the peritoneal cavity, glomerular and tubular alterations in kidneys, and differences in the diameter of the thyroid follicles and thickness of the follicular epithelial cells. The initial days of the reproductive phase revealed MP-related differences in the number of gravid females, fecundity, and fertilization rates. Overall, these values reverted to normal rates throughout the succeeding days. No significant effects of PS MPs exposure were evidenced on offspring success. The 150-day PS MPs dietary exposure used in this study provided clues of histological effects and a reproduction delay. However, it did not seem to compromise overall growth/thriving and the ongoing reproduction.

Effects of life cycle exposure to dietary 2,2', 4,4'-tetrabromodiphenyl ether (BDE-47) on medaka fish (Oryzias latipes).

Previous studies conducted in our laboratory, which resorted to 40-day oral exposures to BDE-47 in specific developmental windows of medaka (Oryzias latipes) did not evidence effects on growing or breeding periods. In this new study, full life cycle (i.e. 140-day) dietary exposure to 1000 ng of BDE-47/g was performed with medaka to evaluate effects on growth and reproduction (i.e. fecundity, fertility, hatchability), and to analyze the bioacumulated BDE-47 in and transferred to offspring. No significant effects were observed for the biometric analyses during the growth and maturation periods and no biased sex ratios were found. Reproductive capacity was not affected by the presence of BDE-47 in diet. There was no evidence for apparent effects from parental exposure during embryo and eleutheroembryo development. The analytical results revealed steady BDE-47 bioaccumulation during the growing period, which remained in the reproductive phase in males, and a decreasing tendency was noted in females. These lowering BDE-47 levels in females coincided with the detected BDE-47 levels offloaded in embryos. In the 10-day-old post-hatch larvae, the BDE-47 concentrations dropped to comparatively lower values than the concentrations detected in parents. This finding suggests an efficient metabolic process in the eleutheroembryonic and post-eleutheroembryonic phases. Our 140-day dietary approach found no BDE-47 effects on medaka growth and reproduction, or in early progeny stages despite effective bioaccumulation and maternal transfer.

Effect assessment of reclaimed waters and carbamazepine exposure on the thyroid axis of Xenopus laevis: Gene expression modifications.

Reclaimed water (RW) obtained from wastewater treatment plants (WWTP) is used for irrigation, groundwater recharge, among other potential uses. Although most pollutants are removed, traces of them are frequently found, which can affect organisms and alter the environment. The presence of a myriad of contaminants in RW makes it a complex mixture with very diverse effects and interactions. A previous study, in which tadpoles were exposed to RW and RW spiked with Carbamazepine (CBZ), presented slight thyroid gland stimulation, as suggested by the development acceleration of tadpoles and histological findings in the gland provoked by RW, regardless of the CBZ concentration. To complement this study, the present work analysed the putative molecular working mechanism by selecting six genes coding for the thyroid-stimulating hormone (TSHß), thyroid hormone metabolising enzymes (DIO2, DIO3), thyroid receptors (THRA, THRB), and a thyroid hormone-induced DNA binding protein (Kfl9). Transcriptional activity was studied by Real-Time PCR (RT-PCR) in brains, hind limbs, and tails on exposure days 1, 7, and 21. No significant differences were observed between treatments for each time point, but slight alterations were noted when the time response was analysed. The obtained results indicate that the effects of RW or RW spiked with CBZ are negligible for the genes analysed during the selected exposure periods.

The effect of PFOs on the uptake and translocation of emerging contaminants by crops cultivated under soil and soilless conditions.

The Mediterranean is a region of substantial agriculture production that faces concurrent environmental stresses and freshwater pollution given the occurrence of emerging contaminants (ECs). Among these pollutants, the surface-active substances have been suggested to enhance the bioavailability of other ECs. This research evaluates a comparative uptake and translocation assessment of irrigation exposure to atenolol (ATN, 60 µg/L), carbamazepine (CBZ, 60 µg/L) and triclosan (TCS, 30 µg/L) alone vs. these combined with perfluorooctanesulfonic acid (PFOS, 10 µg/L) under semifield (i.e., soil experimental set) vs. hydroponics (i.e., soilless experimental set) growing conditions with lettuce, radish and tomato plants. Both experimental sets revealed efficient root uptake and translocation for the three ECs regardless of their co-existence with PFOS. The overall results of the uptake and translocation of the ECs in the lettuce and tomato plants suggested a simultaneous treatment-plant organ interaction, which was not affected by PFOS being present in both experimental sets. PFOS in irrigation water did not increase cellular perviousness to the other three ECs. These observations support the hypothesis of factors other than PFOS being responsible for the differential bioaccumulation and translocation potentials seen in both experimental sets. However, the radish plants co-irrigated with PFOS brought about increased movement of ECs from roots to aerial parts, more specifically ATN and CBZ in the soil experimental set, and ATN and TCS in the soilless set. These results support the notion that factors inherent to the physiological characteristics of this root vegetable contributed to ECs' increased tendency to move from roots to aerial parts. Despite the three ECs efficiently accumulating, the risk to humans from eating the edible parts of these plants grown under soil or soilless conditions was low.

Developmental/reproductive effects and gene expression variations in Chironomus riparius after exposure to reclaimed water and its fortification with carbamazepine and triclosan.

The potential benefits of reclaimed water (RW) uses for environmental enhancement and restoration could become adverse impacts if RW does not meet the quality criteria that ensure wildlife preservation. RW can contain complex mixtures of micropollutants that may accumulate in sediment after environmental uses and affect benthic fauna. Therefore, we designed this study to assess the effects of RW on a sediment insect species used mainly in ecotoxicology (Chironomus riparius). Whole organism effects and gene expression were measured in a water sediment system after spiking RW as overlying water, which was renewed 3 times during the test. Development rate, emergence rate and fecundity were monitored after the 21-day exposure. Endocrine-related genes (EcR, ERR, E75, Vtg), cellular stress genes (hsp70, hsc70, hsp24, hsp10) and biotransformation genes (gp93, GSTd3, GPx, cyp4g) were assessed in larvae after the 10-day exposure. The experimental design also included single or binary fortifications of both test medium and RW, obtained by adding two emerging pollutants: carbamazepine (100 µg/L CBZ) and triclosan (20 µg/L TCS). The chemical characterisation of RW showed that 20 of the 23 screened emerging pollutants fell within the detection limit, 10 exceeded 0.01 µg/L (including CBZ) and three exceeded 0.1 µg/L (hydrochlorothiazide, atenolol, ibuprofen). The analytical measures of sediment (day 21) and overlying water (days 7, 14 and 21) were taken to know the water-sediment distribution of CBZ and TCS added to fortifications. CBZ distributed mainly in overlying water (110-164 µg/L and 73-100 µg/kg), while TCS showed a higher affinity to sediment (2.8-5.1 µg/L and 36-55 µg/kg). RW had significant effects in molecular terms (Vtg, hsp70, hsc70), but had no significant effects on the whole organism. Nevertheless, the single RW fortifications impaired both the development rate and fecundity, while the binary RW fortification impaired only fecundity. The most marked increase in EcR expression was observed for the binary RW fortification. Hsps, GSTd3 and cyp4g showed a similar tendency to that observed for EcR and Vtg in the binary and single RW fortifications. The binary mixture (CBZ and TCS together) in RW was toxic, but not in the medium tests. Therefore, the major concern of RW uses is apparently related to the interactivity between this complex matrix and any other pollutants possibly present in the environment where RW is applied. Our results underscore the need for raising awareness about RW effects, which can be achieved by ecotoxicological testing.

Uptake of atenolol, carbamazepine and triclosan by crops irrigated with reclaimed water in a Mediterranean scenario.

Water scarcity is a natural condition in the Mediterranean rim countries. In this region, reuse of reclaimed water (RW) from wastewater treatment plants (WWTPs) is becoming a potential source for highly water-demanding activities such as agriculture. However, the removal capacity of contaminants in regular WWTPs has been found to be limited. Considering a Mediterranean scenario, this research investigated the plant uptake and translocation of three representative pharmaceuticals and personal care products (PPCPs) typically present in RW samples from a WWTP located in an urban area in Spain, and assessed the potential risk to humans from plant consumption. The RW samples were collected and analyzed for three representative PPCPs (atenolol -ATN-, carbamazepine -CBZ- and triclosan -TCS-). The target contaminants were also spiked at two levels in the RW samples to consider two worst-case scenarios. Three plant models (lettuce, maize and radish) were grown outdoors and irrigated with four treatments: tap water; RW samples, and the two spiked RW samples. Generally speaking, results revealed an efficient root uptake for the three PPCPs regardless of plant species and fortification level, and suggested an interaction effect of treatment and plant organ. Different bioaccumulation and translocation potentials of the three PPCPs were seen into the aerial organs of the plants. Overall, these observations support the idea that factors including the physico-chemical properties of the PPCPs and physiological plant variables, could be responsible for the differential accumulation and translocation potentials observed. These variables could be critical for crops irrigated with RW in regions with extended dry seasons, high solar incidence and low annual rainfall such as those in the Mediterranean rim where plants are subjected to high transpiration rates. However, the results obtained from this experimental approach suggested a negligible risk to humans from consumption of edible plants irrigated with RW samples with presence of PPCPs, despite the fact that the three representative PPCPs under study accumulated efficiently in the plants.

The role of PFOS on triclosan toxicity to two model freshwater organisms.

Surface-active substances may enhance the bioavailability of certain pollutants by modifying the permeability of cell membranes. However, they could also interact in a positive manner by increasing toxicity to aquatic organisms. A comparative effects assessment of waterborne exposure to triclosan (TCS) alone vs. combined with perfluorooctanesulfonic acid (PFOS) was herein investigated in daphnids (Daphnia magna) and medaka (Oryzias latipes) early life stages (ELS) using a battery of non-invasive behavioral, physiological and anatomical endpoints. Additionally, TCS bioaccumulation was evaluated in medaka embryos to help discern differences in effects caused by either, changes in TCS permeability or by a positive interaction with PFOS. The TCS analytical measurements in the medaka ELS exposure media revealed fast dissipation with half-lives < 12 h. The D. magna immobilization and feeding inhibition assays suggested an increased response when TCS (≥200 and 37.50 µg/L, respectively) was co-exposed with PFOS. Concentrations <800 µg TCS/L did not affect medaka ELS. However, exposures to ≥400 µg TCS/L + PFOS had effects on the embryo and eleutheroembryo viability. The morphometric analysis of the embryonic gallbladder area and the oxidative stress, determined in vivo by the manifestation of reactive oxygen species (ROS), revealed a hormetic response in both experimental sets. The TCS + PFOS experimental set generally resulted in increased gallbladder areas and ROS activity levels compared to those quantified in the corresponding TCS set. The bioaccumulation studies in the medaka embryos revealed comparable TCS levels regardless of PFOS presence. Without disregarding any TCS's enhanced bioavailability caused by the surface-active substance, overall results primarily indicate increased biological effects of TCS due to a potentiation action of PFOS as a binary mixture with TCS.

Combining the assessment of apical endpoints and gene expression in the freshwater snail Physa acuta after exposure to reclaimed water.

Post-treatment wastewater reuses are diverse. Recreational and environmental restoration uses of reclaimed water (RW) can be potentially harmful to aquatic organisms. In this work the freshwater snail Physa acuta was exposed to RW (100%) and its dilution (RW 50%). A simple laboratory mixture of three emerging pollutants was used to address the complex problem of mixture toxicity of RW. Hence fortified reclaimed water (FRW), obtained by adding fluoxetine (400 µg FLX/L), perfluorooctane sulphonic acid (90 µg PFOS/L) and methylparaben (9 µg MP/L), was tested at two dilution percentages: 100% and 50%. The effects of the laboratory mixture of FLX, PFOS and MP on the test medium were also studied. Long-lasting effects, together with early molecular responses, were assessed. Fecundity (cumulative egg production) over 21 days and the hatching of produced eggs (F1) after another 21-day embryonic exposure were monitored. The gene expression of three genes was analysed after 24 h of exposure: two endocrine-related nuclear receptors (ERR and RXR) and one stress protein gene (Hsp70). This reproduction test, with additional assessments of the F1 recovered eggs' hatching success, showed that both RW and FRW significantly reduced fecundity. F1 hatching was affected only by FRW. The gene expression results showed that the RXR response was strikingly similar to the fecundity response, which suggests that this nuclear receptor is involved in the reproductive pathways of gastropods. ERR remained virtually unaltered. Hsp70 was overexpressed by the laboratory mixture in the test medium, but no effect was observed in the fortification of RW. This opposite effect and lack of response for F1 hatching produced by the laboratory mixture in the test medium highlighted the difficulty of predicting mixture effects. The experimental approach allowed us to test the effects caused by RW on P. acuta at different biological organisation levels. Thus, the combination of molecular biomarkers and ecological relevant endpoints is a good strategy to test complex mixtures like RW as it provides a framework to link mechanisms of action and whole organism effects when it is almost impossible to detect the pollutant(s) that cause toxic effects.

Stage-dependent effects of chlorpyrifos on medaka (Oryzias latipes) swimming behavior using a miniaturized swim flume.

By considering chlorpyrifos (CPF), an organophosphorus pesticide with known mechanisms of action that affect neurobehavioral development, we assessed the validity and sensitivity of a miniaturized swim flume by investigating the effects of the insecticide on swimming behavior in medaka (Oryzias latipes) fish growing stages. Medaka in three developmental periods, namely 0, 20 and 40 day-old post-hatch (i.e. time points 0, 20 and 40, respectively), were exposed to CPF (12.5, 25, 50 and 100 µg/L) for 48 h under semi-static conditions. The CPF half-lives during exposures were evaluated and the swimming patterns in a flume section (arena) were presented on two-dimensional gradient maps of forced movement of fish against water current. A comparative numerical analysis of fish residence times between each time point control and the corresponding CPF groups was performed by dividing arenas into 15 proportional areas. The time point 0 control group gradient map showed a noticeably different swim pattern from those of the ≥12.5 µg CPF/L groups, which was statistically supported by the differences for residence times seen in ≥12 corresponding areas. The control group gradient maps for time points 20 and 40 differed from those of the respective ≥12.5 µg CPF/L groups. The comparative analysis of the residence times in the corresponding 15 areas revealed differences in ≥5 areas for time point 20 and in ≥3 areas for time point 40. The integrative analysis of the gradient maps and the numerical statistics revealed stage-specific effects and a concentration-response relationship between CPF and alterations on forced medaka swimming despite the dissipation of CPF from the water column. These results indicate the validity of the miniaturized swim flume toward a more environmentally realistic scenario for the evaluation of neurodevelopmental and behavioral toxicity in small fish models.

Effects of dietary 2,2', 4,4'-tetrabromodiphenyl ether (BDE-47) exposure on medaka (Oryzias latipes) swimming behavior.

A diet fortified with 2,2', 4,4'-tetrabromodiphenyl ether (BDE-47: 0, 10, 100, and 1000 ng/g) was dosed to 4-7-day-old post-hatch medaka fish for 40 days to evaluate the effects on the swimming activity of fish using a miniaturized swimming flume. Chlorpyrifos (CF)-exposed fish were selected as the positive control to assess the validity and sensitivity of the behavioral findings. After 20 and 40 days of exposure, the locomotor activity was analyzed for 6 min in a flume section (arena). The CF positive control for each time point were fish exposed to 50 ng CF/ml for 48 h. Swimming patterns, presented as two-dimensional heat maps of fish movement and positioning, were obtained by geostatistical analyses. The heat maps of the control groups at time point 20 revealed visually comparable swimming patterns to those of the BDE-47-treated groups. For the comparative fish positioning analysis, both the arenas were divided into 15 proportional areas. No statistical differences were found between residence times in the areas from the control groups and those from the BDE-47-treated groups. At time point 40, the heat map overall patterns of the control groups differed visually from that of the 100-ng BDE-47/g-treated group, but a comparative analysis of the residence times in the corresponding 15 areas did not reveal consistent differences. The relative distances traveled by the control and treated groups at time points 20 and 40 were also comparable. The heat maps of CF-treated fish at both time points showed contrasting swim patterns with respect to those of the controls. These differential patterns were statistically supported with differences in the residence times for different areas. The relative distances traveled by the CF-treated fish were also significantly shorter. These results confirm the validity of the experimental design and indicate that a dietary BDE-47 exposure does not affect forced swimming in medaka at growing stages.

Bioaccumulation, maternal transfer and effects of dietary 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) exposure on medaka fish (Oryzias latipes) offspring.

A previous study conducted in our laboratory with growing medaka (Oryzias latipes) showed the capacity of BDE-47 (10-1000ng/g) to bioaccumulate during a 40-day oral exposure. However, the results did not provide evidence for effects during or after the exposure period. In this study, breeding medakas were fed a diet for 40days that contained 1000ng of BDE-47/g. At predefined time points, females (time points 10, 20, 30 and 40), males (time points 30 and 40) and pools of laid eggs (time points 10, 20, 30 and 40) were sampled and collected for: 1) the BDE-47 quantitative analysis in adults in the <24-h-old post-fertilization (hpf) embryos, and in the <24-h-old post-hatch (hph) eleutheroembryos; 2) the evaluation of fecundity, fertility and hatching. Additional pools of embryos collected at time point 40 were evaluated for: 1) the active swimming behavior of the 48hph offspring in the eleutheroembryonic stage; 2) the BDE-47 quantification in the 240hph resultant larvae. BDE-47 accumulated in parents rapidly, and concentrations remained constant at higher levels in males (values within the 50-60ng/g wet weight -w.w.- range) compared with females (70ng/g w.w. range). The BDE-47 concentrations detected in embryos and eleutheroembryos ranged from 200 to 500ng/g w.w. for time points 10-40. Reproductive capacity, hatching and ensuing swim bladder inflation were not affected by parental BDE-47 dietary exposure, nor was the active swimming behavior in eleutheroembryos. The BDE-47 concentration in the 240hph larvae lowered to levels close to those detected in parents. Despite the efficient BDE-47 maternal transfer, these results offered no evidence for BDE-47 effects on fish reproduction or in the early life stages of offspring.

Effects of dietary 2,2', 4,4'-tetrabromodiphenyl ether (BDE-47) exposure in growing medaka fish (Oryzias latipes).

In this research work, we addressed the effects of a diet fortified with BDE-47 (0, 10, 100, 1000ng/g) dosed to 4-7 day-old post-hatch medaka fish for 40 days, followed by an 80-day depuration period. BDE-47 accumulation and overall growth were evaluated throughout the dosing period, and its elimination was quantified over the following 60 days. The histological condition of the thyroid gland, liver and gonads from the 1000ng BDE-47-treated fish were assessed 5 and 70days after exposures finished. The phenotypic males to females ratio was also quantified 70days after treatments finished. Sixty days after the BDE-47 exposures, reproductive capacity (i.e. fecundity, fertility and hatchability) was evaluated in mating groups for a 20-day period. BDE-47 exposure via food from larval through juvenile life stages of medaka fish resulted in steady accumulation with time dose-dependently. This accumulation tendency rapidly decreased after dosing ended. The growth rates showed a significant increase only at the highest concentration 70days after exposures finished. The histological survey did not reveal BDE-47-related alterations in the condition of the potential target organs. However, a morphometrical approach suggested BDE-47-related differences in the thickness of the epithelium that lines thyroid follicles. The reproduction studies showed comparable values for the fecundity, fertility and hatching rates. Dietary BDE-47 dosed for 40days to growing medaka fish did not alter the phenotypic sex ratios at maturity. The dietary approach used herein could not provide conclusive evidence of effects on medaka development and thriving despite the fact that BDE-47 underwent rapid accumulation in whole fish during the 40-day treatment.

Limitations of waterborne exposure of fish early life stages to BDE-47.

2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is acknowledged as the most abundant congener of all polybrominated diphenyl ethers (PBDEs). Despite its limited residence in the water column, most ecotoxicological research using fish early life stages (ELS) has focused on its waterborne bioavailability. These studies have been supported either by chemical analysis in solutions or in tissues after ≤ 168 h exposures to relatively high waterborne concentrations with dimethyl sulfoxide (DMSO) as solvent carrier (≤ 0.5%). Using noninvasive physiological and anatomical features in medaka ELS, we investigated the viability of waterborne BDE-47 exposures (100-10,000 µg/L; 1% DMSO) and evaluated the developmental effects in relation to the actual BDE-47 present in water. Embryos were exposed for 10 days under semi-static (24-h renewal) conditions and waterborne BDE-47 concentrations (i.e., dissolved) were quantitated daily and their accumulation in eleutheroembryonic tissues was analyzed 4 days after exposures finished. BDE-47 in solution rapidly decreased after each renewal by >50% in 24h. This was confirmed by discernible precipitation occurring at ≥ 5,000 µg/L on the bottom of the container and attached to the chorionic filaments of eggshell. The fast dissipation from water may explain why, besides the subtle, yet significant effects on post-hatching growth (short length at ≥5000µg/L), no other significant deleterious developmental effects were observed despite the fact that BDE-47 accumulated in tissues in response to BDE-47 treatment. Waterborne BDE-47 exposure was unachievable under traditional semi-static exposure conditions, but was achievable in repeated pulse exposures lasting a few hours whenever the medium was renewed. Hence, this research encourages the use of alternate - more realistic - exposure routes (e.g., particulate matter or sediments) when evaluating early developmental toxicity of BDE-47 or any other PBDE sharing similar properties.