Thyroid-like hormone signaling in invertebrates and its potential role in initial screening of thyroid hormone system disrupting chemicals.

This review examines the presence and evolution of thyroid-like systems in selected aquatic invertebrates to determine the potential use of these organisms in screens for vertebrate thyroid hormone axis disrupting chemicals (THADCs). Such a screen might support the phasing out of some vertebrate testing. Although arthropods including crustaceans do not contain a functional thyroid signaling system, elements of such a system exist in the aquatic phyla mollusks, echinoderms, tunicates, and cephalochordates. These phyla can synthesize thyroid hormone, which has been demonstrated in some groups to induce the nuclear thyroid hormone receptor (THR). Thyroid hormone may act in these phyla through interaction with a membrane integrin receptor. Thyroid hormone regulates inter alia metamorphosis but, unlike in vertebrates, this does not occur via receptor activation by the ligands triiodothyronine (T3) and thyroxine (T4). Instead, the unliganded nuclear receptor itself controls metamorphosis in mollusks, echinoderms, and tunicates, whereas the T3 derivative tri-iodothyroacetic acid (TRIAC) acts as a THR ligand in cephalochordates. In view of this, it may be possible to develop an invertebrate-based screen that is sensitive to vertebrate THADCs that interfere with thyroid hormone synthesis or metabolism along with interaction with membrane receptors. The review makes some recommendations for the need to develop an appropriate test method. Integr Environ Assess Manag 2023;19:63-82. © 2022 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Organotins in a food web from the Three Gorges Reservoir, China: Trophic enrichment and potential health risk.

Triphenyltin (TPhT) and tributyltin (TBT) remain widely present in various aquatic environments despite restrictions on their use in many countries for many years. The biomagnification of these compounds in the aquatic food web remains controversial. This study reports the bioaccumulation of TPhT and TBT in aquatic animals in the Three Gorges Reservoir (TGR), a deep-water river channel-type reservoir and the largest reservoir in China. We measured TPhT, TBT and their metabolites in 2 invertebrates, 27 fish and the aquatic environment. The logarithmic bioaccumulation factors of TPhT and TBT were 4.37 and 3.77, respectively, indicating that TPhT and TBT were enriched in organisms of the TGR. Both TPhT and TBT concentrations were significantly and positively correlated with trophic level, with trophic magnification factors of 3.71 and 3.63, respectively, indicating that TPhT and TBT exhibited similar trophic enrichment in the freshwater food web of the TGR. The results of health risk assessment showed that although all hazard index (HI) values were <1, more attention should be paid to the health risk to children associated with consumption of aquatic products (HI = 0.67). This study provides powerful evidence of trophic enrichment of TPhT and TBT in a freshwater food web in a deep-water river channel-type reservoir and provides valuable data regarding organotins in aquatic animals in the TGR.

Ignored microplastic sources from plastic bottle recycling.

The recovery and recycling of plastic products has increased dramatically in recent years as a strategy to achieve sustainable production and minimization of plastic pollution. However, the release of microplastics during plastic recycling has received little attention. We evaluated the generation and fate of microplastics in three typical facilities which make polyethylene terephthalate (PET) flakes using post-consumer PET bottles as raw material. Microplastics, 0.1- 5.0 mm in size, were detected in production wastewater at concentrations ranging from 23.43 ± 1.04 mg/L to 1836.37 ± 31.73 mg/L, while decreased to (8.13 ± 0.42-83.83 ± 0.93) mg/L in discharge effluent and (52,166 ± 2858-68,866 ± 2500) µg/g in sludge. Interestingly, the profiles of microplastics in samples from production wastewater, effluents, and sludge showed significant differences. Although, in all three compartments, the mass of microplastics increased, and the particle number decreased with increasing particle size. Overall, the removal ratio of total microplastics from the production wastewater was 53.47 ± 4.48% to 99.56 ± 0.02% in mass, and from 90.08 ± 0.82% to 99.56 ± 0.05% in quantity. The loss of microplastics from wastewater resulted in their concentration in sludge. Factors that influence the transfer of microplastics from wastewater to sludge should be identified and utilized to maintain a high level of removal and prevent leakage of these particles into the environment.

A proposed nomenclature for microplastic contaminants.

Microplastics are emerging contaminants with a wide environmental distribution and potential to elicit adverse impacts on organisms. Despite this lack of consistency among reports, data obtained from different investigations are often compared, resulting in the potential for misrepresentation of global microplastic contamination. Major interlaboratory variability in quantification of microplastic levels stem from size-related differences in sampling and analysis with different density solutions to separate microplastics. Herein, we propose a nomenclature that provides key information relating to the microplastics abundance in samples. That is, the proposed nomenclature, MPsca, b, informs on mesh or filter size used in sampling, the density of flotation solution used to separate the microplastics, and the detection limit during the analysis progress of microplastics. This proposed nomenclature would facilitate comparisons among studies to avoid over- or under-estimation of global microplastic levels. Moreover, it would also facilitate the interpretation of meta-data in future assessments.

A Crab Is Not a Fish: Unique Aspects of the Crustacean Endocrine System and Considerations for Endocrine Toxicology.

Crustaceans-and arthropods in general-exhibit many unique aspects to their physiology. These include the requirement to moult (ecdysis) in order to grow and reproduce, the ability to change color, and multiple strategies for sexual differentiation. Accordingly, the endocrine regulation of these processes involves hormones, receptors, and enzymes that differ from those utilized by vertebrates and other non-arthropod invertebrates. As a result, environmental chemicals known to disrupt endocrine processes in vertebrates are often not endocrine disruptors in crustaceans; while, chemicals that disrupt endocrine processes in crustaceans are often not endocrine disruptors in vertebrates. In this review, we present an overview of the evolution of the endocrine system of crustaceans, highlight endocrine endpoints known to be a target of disruption by chemicals, and identify other components of endocrine signaling that may prove to be targets of disruption. This review highlights that crustaceans need to be evaluated for endocrine disruption with consideration of their unique endocrine system and not with consideration of the endocrine system of vertebrates.

Endocrine Disruption in Invertebrates: A Survey of Research Progress.

Manmade chemicals can interfere with endocrine processes and have permeated many ecosystems. Arguably, the most devastating example of endocrine disruption occurred in gastropod molluscs which led to the banning of tributyltin. The invertebrates consist of ∼95% of all known animals and possess endocrine systems that can significantly differ from that of vertebrates. An expert group in the late 1990s highlighted considerable paucity in our knowledge of these endocrine systems and the limited ability to ascertain risks of endocrine-disrupting chemicals (EDCs) to invertebrates. Twenty years later, we surveyed experts in this field on the current state of the science. Respondents agreed that endocrine disruption is still a significant issue and noted that there was key evidence that EDCs were impacting invertebrates groups. Respondents noted a variety of impediments to advancing the science, including inadequate funding, insufficient knowledge to develop appropriate assays, and generally low support for invertebrate studies. Several scientists highlighted that resources were being misdirected with studies that address impacts of vertebrate EDCs or using biomarkers specific to vertebrate endocrine disruption. Sadly, many of the recommendations proposed by respondents matched those made over two decades ago. Accordingly, the field has not advanced as much as one might have expected.

An effective method for evaluation of microplastic contaminant in gastropod from Taihu Lake, China.

Microplastics are ubiquitous in the environment. The isolation and characterization of microplastics can change, enabling science to elucidate the fate of microplastics in organisms. The main objective of the present study was to develop a rapid and effective method for the isolation, characterization, and quantification of microplastics from gastropod, and then evaluate the microplastic pollution in wild freshwater snails using the developed method. The whole tissue of gastropod Bellamya aeruginosa was spiked with microplastics derived from cosmetic products to optimize the tissue digestion and microplastic identification process. Optimum digestion of soft tissue was performed using a mixture of Tris-HCl, proteinase K, and KOH. Recovery of microplastics from the tissue digests, as determined by microscopy and infrared spectroscopy, was 89 ± 5%. The entire procedure could be completed within 30 h. Application of the procedure to wild freshwater snail B. aeruginosa collected from Taihu Lake revealed that 90~100% of the sampled snails accumulated 1 to 4 types of microplastics including poly(vinyl acetate), polyethylene terephthalate, polystyrene, and polyamides. In summary, a quick method was developed for the isolation and identification of microplastics from gastropod tissues, and the application of the method revealed the presence of microplastics in snails inhabiting Taihu Lake, China.

Investigation of the microplastics profile in sludge from China's largest Water reclamation plant using a feasible isolation device.

Sewage sludge, which is widely applied to land as a fertilizer, is a key source of microplastics in the environment. We sought to develop a feasible device for isolation of microplastic from sewage sludge for further understanding their fates in the environment. In the present study, an effective isolation device, consisting of a fritted glass funnel and a glass filtration apparatus, was constructed to extract microplastics from sludge with nearly 100% recovery efficiency. Then, a high abundance of microplastics was detected in sludge sampled from China's largest sewage treatment plant. Among the 25 types of microplastic polymers confirmed by Fourier transform infrared spectroscopy, poly(11-bromoundecyl acrylate) (PBA) and poly(11-bromoundecyl methacrylate) (PBMA) accounted for 23.63% of total microplastics detected. Rayon, polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), and copolymers, such as PP/PE and poly(styrene:acrylonitrile:butadiene) (ABS), were also detected. The shapes of these microplastics consisted of pellets, fragments, films, and microfibers. Characterization of the isolated microplastics revealed that domestic applications and vehicle products were the major sources of microplastic in sewage treatment sludge. Some priority recommendations were issued based on these results. In conclusion, the present study demonstrate that the device is effective for the isolation of microplastics from sludge.

Involvement of glutamate and serotonin transmitter systems in male sex determination in Daphnia pulex.

Environmental sex determination occurs in many organisms, however the means by which environmental stimuli are translated into endocrine messages remains poorly understood. The N-methyl-ᴅ-aspartate receptor (NMDAR) was evaluated as a candidate neural sensor of environmental signals linking environmental cues to endocrine responses using the crustacean Daphnia pulex. NMDAR agonists, modulators, and antagonists were evaluated for their ability to impact D. pulex male sex determination during early stages of reproductive maturity under conditions that simulated seasonal change. The antagonists MK-801 and desipramine significantly increased male sex determination. Both chemicals are also modulators of serotonergic and noradrenergic systems, thus, we evaluated several modulators of monoamine neurotransmission in an effort to discern which signaling pathways might contribute to male sex determination. Compounds that altered serotonergic signaling also stimulated male sex determination. The involvement of the glutamate and monoamine signaling in male sex determination was supported by the increase in mRNA levels of related receptors and transporters under conditions that stimulate male sex determination. Further, mRNA levels of components of the terminal endocrine pathway responsible for male sex determination were also elevated under stimulatory conditions. Overall, we provide evidence that glutamatergic and serotonergic systems function upstream of the endocrine regulation of male sex determination in early life stage daphnids.

Phthalate Esters and Their Potential Risk in PET Bottled Water Stored under Common Conditions.

A great deal of attention has been paid lately to release of phthalate esters (PAEs) from polyethylene terephthalate (PET) bottles into PET bottled drinking water due to their potential endocrine-disrupting effects. Three kinds of PAEs, including diethyl phthalate (DEP), dimethyl phthalate (DMP) and dibutyl phthalate (DBP), were detected in 10 popular brands of PET bottles in Beijing, ranging from 101.97 µg/kg to 709.87 µg/kg. Meanwhile, six kinds of PAEs, including DEP, DMP, DBP, n-butyl benzyl phthalate (BBP), di-n-octyl phthalate (DOP) and di(2-ethylhexyl) phthalate (DEHP), were detected in PET bottled water, ranging from 0.19 µg/L to 0.98 µg/L, under an outdoor storage condition, while their concentrations ranged from 0.18 µg/L to 0.71 µg/L under an indoor storage condition. Furthermore, the concentrations of PAEs in brand D and E bottles were slightly increased when the storage time was prolonged. In addition, the concentrations of PAEs in commercial water contained in brand B and H bottles and pure water contained in brand E and G bottles were also slightly increased with the increase of storage temperature. Interestingly, DBP mainly contributed to the increased PAEs levels in simulation water. These results suggest that a part of the PAEs in PET bottled water originated from plastic bottles, which was related to the storage time and temperature. However, the PAEs in PET bottled water only pose a negligible risk to consumers if they follow the recommendations, such as storage at a common place (24 °C), away from sun and in a short period of time.

The role of nuclear receptor E75 in regulating the molt cycle of Daphnia magna and consequences of its disruption.

Biological rhythms regulate innumerable physiological processes, yet little is known of factors that regulate many of these rhythms. Disruption in the timing of these rhythms can have devastating impacts on population sustainability. We hypothesized that the timing of the molt infradian rhythm in the crustacean Daphnia magna is regulated by the joint action of the protein E75 and nitric oxide. Further, we hypothesized that disruption of the function of E75 would adversely impact several physiological processes related to growth and reproduction. Analysis of mRNA levels of several genes, involved in regulating the molt cycle in insects, revealed the sequential accumulation of E75, its dimer partner HR3, FTZ-F1, and CYP18a1 during the molt cycle. Exposure to the nitric oxide donor sodium nitroprusside early in the molt cycle had no effect on E75 or HR3 mRNA levels, but delayed the peak accumulation of FTZ-F1 and CYP18a1 mRNA. The subsequent exuviation was also delayed consistent with the delay in peak accumulation of FTZ-F1 and CYP18a1. These results supported our assertion that nitric oxide binds E75 rendering it incapable of binding HR3. Excess HR3 protein then enhanced the accumulation of the downstream products FTZ-F1 and CYP18a1. Similarly, suppression of E75 mRNA levels, using siRNA, had no effect on mRNA levels of HR3 but elevated mRNA levels of FTZ-F1. Consistent with these molecular responses, the suppression of E75 using siRNA increased the duration of the molt cycle and reduced the number of offspring produced. We conclude that the molt cycle of daphnids is regulated in a manner similar to insects and disruption of E75 results in a lengthening of the molt cycle and a reduction the release of viable offspring.

Complementary roles of photoperiod and temperature in environmental sex determination in Daphnia spp.

Daphnia spp., a keystone genus in freshwater lentic habitats, are subject to environmental sex determination wherein environmental conditions dictate offspring sex and whether they reproduce asexually or sexually. The introduction of males into a population denotes the first step in the switch from asexual parthenogenetic reproduction to sexual reproduction. We tested the hypothesis that photoperiod and temperature co-regulate male sex determination and that these environmental stimuli would activate elements of the male sex determination signaling cascade. The results revealed that photoperiod was a critical cue in creating permissive conditions for male production. Further, under photoperiod-induced permissive conditions, male sex determination was temperature dependent. The two daphnid species evaluated, Daphnia pulex and Daphnia magna, exhibited different temperature dependencies. Daphnia pulex produced fewer males with increasing temperatures between 16 and 22°C, and D. magna exhibited the opposite trend. We found consistent expression patterns of key genes along the male sex-determining signaling pathway in D. pulex independent of environmental stimuli. mRNA levels for the enzyme responsible for synthesis of the male sex-determining hormone, methyl farnesoate, were elevated early in the reproductive cycle, followed by increased mRNA levels of the methyl farnesoate receptor subunits Met and SRC Environmental conditions that stimulated male offspring production significantly increased Met mRNA levels. The results indicate that male sex determination in daphnids is under the permissive control of photoperiod and the regulatory control of temperature. Further, these environmental cues may stimulate male sex determination by increasing levels of the Met subunit of the methyl farnesoate receptor.

Freshwater snail Parafossarulus striatulus estrogen receptor: Characteristics and expression profiles under lab and field exposure.

The modes of action by which putative endocrine disrupting chemicals (EDCs) elicit toxicity in mollusks remains unclear due to our limited understanding of the molluscan endocrine system. We identified and partially characterised the estrogen receptor (ER) of the mollusk Parafossarulus striatulus. The full-length cDNA of the ER of P. striatulus (psER) was isolated and found to have an ORF of 1386 bp which corresponded to 461 amino acids. Phylogenetic analysis revealed that psER is an orthologue of ER of other mollusks. Moreover, the DNA-binding domain, ligand-binding domain, P-box, D-box, and AF2 domain were also identified in psER. Exposure of females and males to 17ß-estradiol (E2, 100 ng/L) for 24 h and 72 h did not alter psER transcription, but exposure to 17α-methyltestosterone (MT, 100 µg/L) for 72 h significantly decreased ER transcription in females only (p < 0.05). psER transcription was surveyed in males and females seeded in different regions in Taihu Lake, China. psER transcription were elevated among females and males maintained at site ML. This elevation was statistically significant (p < 0.05) among male snails as compared to snails held at the more pristine site of SZ. This was different to the results from lab, implying that some unknown chemicals or other environmental factors in field could affect psER transcription level in snails. Furthermore, females and males held at site ML also exhibited a significant elevation in vitellogenin transcription as compared to snails held at site SZ, suggesting that vitellogenin production may be directly regulated by psER or co-regulated with psER in this species.

No impacts of microcystins on wild freshwater snail Bellamya Aeruginosa fecundity from a eutrophic lake.

The preliminary investigation at shoreline along Taihu lake with different degrees of eutrophication status found no significant relationship between the microcystin-LR concentrations and the freshwater snail Bellamya aeruginosa fecundity or the abundance of wild freshwater snails. To further confirm the impact of eutrophication on the reproductive ability of snails, ecological mesocosm experiments were employed at four sites in Taihu lake during the algal blooming period, and no significant relationship was also found between MC-LR concentrations and snail fecundity. These results implied that eutrophication does not negatively or positive affect snail fecundity in Taihu Lake, a typical eutrophication lake in China.

Hemoglobin Levels Modulate Nitrite Toxicity to Daphnia magna.

Nitrogenous compounds enter the environment through various anthropogenic sources. Among these are nitrate (NO3-) and nitrite (NO2-) which can oxidize the heme moiety of hemoglobin and reduce the oxygen-carrying capacity of the molecule resulting in toxicity. Of the two anions, nitrite is more toxic. Hemoglobin levels are influenced by environmental conditions; thus, we hypothesized that hemoglobin levels would influence the toxicity of nitrite with low hemoglobin levels resulting in enhanced toxicity and high hemoglobin levels resulting in reduced toxicity. We tested this hypothesis by elevating hemoglobin levels with pyriproxyfen treatment and lowering hemoglobin levels using siRNA in Daphnia magna. Exposure to pyriproxyfen significantly elevated hemoglobin mRNA levels and induced copper coloration of the organisms, indicative of increased hemoglobin protein accumulation. siRNA treatment significantly reduced hemoglobin mRNA levels in both untreated and pyriproxyfen-treated organisms and attenuated copper coloration. Pyriproxyfen treatment increased the tolerance of daphnids to the acute toxicity of nitrite approximately 2-fold while siRNA treatment significantly decreased the tolerance of daphnids to nitrite toxicity. Results indicate that increased hemoglobin levels increase the tolerance of daphnids to nitrite toxicity which may serve to protect daphnids in environments subject to hemoglobin-elevating hypoxia or elevated temperatures.

Continuous Structured Population Models for Daphnia magna.

We continue our efforts in modeling Daphnia magna, a species of water flea, by proposing a continuously structured population model incorporating density-dependent and density-independent fecundity and mortality rates. We collected new individual-level data to parameterize the individual demographics relating food availability and individual daphnid growth. Our model is fit to experimental data using the generalized least-squares framework, and we use cross-validation and Akaike Information Criteria to select hyper-parameters. We present our confidence intervals on parameter estimates.

Ligand-Mediated Receptor Assembly as an End Point for High-Throughput Chemical Toxicity Screening.

The high throughput screening of chemicals for interaction with intracellular targets is gaining prominence in the toxicity evaluation of environmental chemicals. We describe ligand-mediated receptor assembly as an early event in receptor signaling and its application to the screening of chemicals for interaction with targeted receptors. We utilized bioluminescence resonance energy transfer (BRET) to detect and quantify assembly of the methyl farnesoate receptor (MfR) in response to various high-production volume and other chemicals. The hormone methyl farnesoate binds to the MfR to regulate various aspects of reproduction and development in crustaceans. The MfR protein subunits Met and SRC, cloned from Daphnia pulex, were fused to the fluorophore, mAmetrine and the photon generator, Rluc2, respectively. Ligand-mediated receptor assembly was measured by photon transfer from the photon donor to the fluorophore resulting in fluorescence emission. Overall, the BRET assay had comparable or greater sensitivity as compared to a traditional reporter gene assay. Further, chemicals that screened positive in the BRET assay also stimulated phenotypic outcomes in daphnids that result from MfR signaling. We concluded the BRET assay is an accurate, sensitive, and cost/time efficient alternative to traditional screening assays.

Preliminary evidence for snail deformation from a Eutrophic lake.

The incidence of deformities in snails Bellamya aeruginosa was investigated in a typical eutrophicated lake - Taihu Lake. A total of 15 105 specimens were collected, and 0.18-0.93% of the snails exhibited abnormal tentacle bifurcations. Abnormally developed snails were all female and were found in regions with relatively high Chlorophyll a levels (12.40±7.23µg/L). As tentacles are sexually dimorphic in B. aeruginosa, we postulated that factors associated with eutrophication might be responsible for the partial masculinization of tentacles in females. Differential gene expression analyses revealed that a number of unigenes were significantly up-regulated or down-regulated in snails sampled from three locations having high Chlorophyll a levels compared with snails sampled from the region with lower Chlorophyll a level (2.95µg/L). Thus, transcriptomic profiling revealed potential molecular signal of eutrophication that can lead to developmental abnormalities in this species.

Agonist-mediated assembly of the crustacean methyl farnesoate receptor.

The methyl farnesoate receptor (MfR) orchestrates aspects of reproduction and development such as male sex determination in branchiopod crustaceans. Phenotypic endpoints regulated by the receptor have been well-documented, but molecular interactions involved in receptor activation remain elusive. We hypothesized that the MfR subunits, methoprene-tolerant transcription factor (Met) and steroid receptor coactivator (SRC), would be expressed coincident with the timing of sex programming of developing oocytes by methyl farnesoate in daphnids. We also hypothesized that methyl farnesoate activates MfR assembly. Met mRNA was expressed rhythmically during the reproductive cycle, with peak mRNA accumulation just prior period of oocytes programming of sex. Further, we revealed evidence that Met proteins self-associate in the absence of methyl farnesoate, and that the presence of methyl farnesoate stimulates dissociation of Met multimers with subsequent association with SRC. Results demonstrated that the Met subunit is highly dynamic in controlling the action of methyl farnesoate through temporal variation in its expression and availability for receptor assembly.

Validation of a two-generational reproduction test in Daphnia magna: An interlaboratory exercise.

Effects observed within one generation disregard potential detrimental effects that may appear across generations. Previously we have developed a two generation Daphnia magna reproduction test using the OECD TG 211 protocol with a few amendments, including initiating the second generation with third brood neonates produced from first generation individuals. Here we showed the results of an inter-laboratory calibration exercise among 12 partners that aimed to test the robustness and consistency of a two generation Daphnia magna reproduction test. Pyperonyl butoxide (PBO) was used as a test compound. Following experiments, PBO residues were determined by TQD-LC/MS/MS. Chemical analysis denoted minor deviations of measured PBO concentrations in freshly prepared and old test solutions and between real and nominal concentrations in all labs. Other test conditions (water, food, D. magna clone, type of test vessel) varied across partners as allowed in the OECD test guidelines. Cumulative fecundity and intrinsic population growth rates (r) were used to estimate "No observed effect concentrations "NOEC using the solvent control as the control treatment. EC10 and EC-50 values were obtained regression analyses. Eleven of the twelve labs succeeded in meeting the OECD criteria of producing >60 offspring per female in control treatments during 21days in each of the two consecutive generations. Analysis of variance partitioning of cumulative fecundity indicated a relatively good performance of most labs with most of the variance accounted for by PBO (56.4%) and PBO by interlaboratory interactions (20.2%), with multigenerational effects within and across PBO concentrations explaining about 6% of the variance. EC50 values for reproduction and population growth rates were on average 16.6 and 20.8% lower among second generation individuals, respectively. In summary these results suggest that the proposed assay is reproducible but cumulative toxicity in the second generation cannot reliably be detected with this assay.