Effect-based methods including cell-based bioassays, reporter gene assays and whole-organism assays have been applied for decades in water quality monitoring and testing of enriched solid-phase ...extracts. There is no common EU-wide agreement on what level of bioassay response in water extracts is acceptable. At present, bioassay results are only benchmarked against each other but not against a consented measure of chemical water quality. The EU environmental quality standards (EQS) differentiate between acceptable and unacceptable surface water concentrations for individual chemicals but cannot capture the thousands of chemicals in water and their biological action as mixtures. We developed a method that reads across from existing EQS and includes additional mixture considerations with the goal that the derived effect-based trigger values (EBT) indicate acceptable risk for complex mixtures as they occur in surface water. Advantages and limitations of various approaches to read across from EQS are discussed and distilled to an algorithm that translates EQS into their corresponding bioanalytical equivalent concentrations (BEQ). The proposed EBT derivation method was applied to 48 in vitro bioassays with 32 of them having sufficient information to yield preliminary EBTs. To assess the practicability and robustness of the proposed approach, we compared the tentative EBTs with observed environmental effects. The proposed method only gives guidance on how to derive EBTs but does not propose final EBTs for implementation. The EBTs for some bioassays such as those for estrogenicity are already mature and could be implemented into regulation in the near future, while for others it will still take a few iterations until we can be confident of the power of the proposed EBTs to differentiate good from poor water quality with respect to chemical contamination.
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•Effect-based triggers (EBTs) for bioassays discriminate good from poor water quality.•EBTs can be derived by read across from existing water quality guideline values.•Mixture factor warranted for bioassays responding to many different chemicals.•EBT derivation method applicable to every bioassay subject to data availability•Here we derived preliminary EBTs for 32 bioassays and discuss many more.
•All PFASs here tested affected the T4 binding to TTR, an important plasma thyroid hormone transport protein.•In vitro toxicity potency factors were established for thyroid hormone disruption ...potential using the novel TTR-TRβ CALUX® bioassay for major PFASs.•Similar effect-based trigger values by applying in vitro and in vivo potency factors were observed for water (3.0 vs. 2.9 to 4.6 μg PFOA-EQ/L).•AFFF surfactants and CMS (with and without TOP assay) did show similar PFOA-EQ levels by chemical and biological analysis.
Over the last decade, per- and polyfluoroalkyl substances (PFASs) have become one of the most heavily investigated persistent organohalogen compound class of environmental concern. However, knowledge about their toxicology is still scarce, although PFASs as individual compounds and their industrial mixtures were shown to exert effects on the thyroid hormone system.
In vitro toxicity potency factors were established for thyroid hormone transport disruption potential using the novel TTR-TRβ CALUX® bioassay for major PFASs. We assessed technical PFASs mixtures, including aqueous film-forming foam (AFFF) surfactants and chromium mist suppressants (CMS) applications with and without total oxidizable precursor (TOP) by TTR-TRβ CALUX® assay for their thyroid hormone transport disrupting potential.
All PFASs listed in the German guideline for drinking water (German Environment Agency, 2017) affected the T4 binding to TTR, an important plasma thyroid hormone transport protein. For all tested PFASs, potency factors based on PC80 values relative to PFOA could be obtained and ranged between PFBA (0.0018) and PFOS (2.0). Applying in vitro potency factors obtained from the present in vitro TTR-TRβ CALUX® assay study and recently reported in vivo potency factors (Zeilmaker et al., 2018; Bil et al., 2021) on the above-mentioned German guideline for PFAS in drinking water, showed that the cumulative effect-based trigger values (in vivo and in vitro) are comparable (3.0 vs. 2.9 to 4.6 μg PFOA-EQ/l). Additionally, AFFF surfactants and CMS with and without TOP assay were tested. Highest activities were found in the older AFFF surfactants (2013/2014) due to higher PFOS/PFOA levels, which were already substituted with 6:2 FTS in 2019, resulting in much lower PFOA-EQ levels. As expected also the PFOA-EQ levels increased in the samples with TOP treatment compared to the original AFFF surfactants and CMS as confirmed here by biological and chemical PFOA-equivalents (PFOA-EQ) analysis.
Additionally, CMS (which have been used in the electroplating chromium industry since the 1950s) as well as PFOS-free, but not PFAS-free fume suppressants (such as Fumetrol® 21) have been tested in the TTR-TRβ CALUX® assay and showed much lower activity levels then the AFFFs, confirmed by the similar potency determination based on chemical PFASs analysis followed by transformation to PFOA-EQ for comparison. The potency factor of 6:2 FTS, which is the main substitute for PFOS in CMS, indicates that it is approximately 100-times less potent as a thyroid hormone disruptor as compared to PFOS.
Potency factors based on PC80 values from TTR-TRβ CALUX® relative to PFOA have been developed for major PFASs. In AFFF surfactants and CMS a trend of higher activities with higher amounts of PFOS and PFOA have been found. PFOA and PFOS showed high responses in the TTR-TRβ CALUX® assay and had the largest contributions to the PFOA-EQs in the AFFF surfactants and CMS applications. Using potency factors as determined in the TTR-TRβ CALUX® to convert PFASs assessed by chemical analysis to PFOA-EQ led to comparable results as compared to the results from PFASs measured directly by the TTR-TRβ CALUX® assay. This study supports the claim that semiquantitative effect- and group-based in vitro CALUX bioanalysis tools can be applied effectively to assess industrial products containing complex mixtures with PFAS compounds for which no instrumental analysis are established, and for many compounds where in vitro toxicity data are not yet available.
Over the last few years, increasing evidence has become available that some brominated flame retardants (BFRs) may have endocrine-disrupting (ED) potencies. The goal of the current study was to ...perform a systematic in vitro screening of the ED potencies of BFRs (1) to elucidate possible modes of action of BFRs in man and wildlife and (2) to classify BFRs with similar profiles of ED potencies. A test set of 27 individual BFRs were selected, consisting of 19 polybrominated diphenyl ether congeners, tetrabromobisphenol-A, hexabromocyclododecane, 2,4,6-tribromophenol, ortho-hydroxylated brominated diphenyl ether 47, and tetrabromobisphenol-A–bis(2,3)dibromopropyl ether. All BFRs were tested for their potency to interact with the arylhydrocarbon receptor, androgen receptor (AR), progesterone receptor (PR), and estrogen receptor. In addition, all BFRs were tested for their potency to inhibit estradiol (sulfation by estradiol sulfotransferase (E2SULT), to interfere with thyroid hormone 3,3′,5-triiodothyronine (T3)–mediated cell proliferation, and to compete with T3-precursor thyroxine for binding to the plasma transport protein transthyretin (TTR). The results of the in vitro screening indicated that BFRs have ED potencies, some of which had not or only marginally been described before (AR antagonism, PR antagonism, E2SULT inhibition, and potentiation of T3-mediated effects). For some BFRs, the potency to induce AR antagonism, E2SULT inhibition, and TTR competition was higher than for natural ligands or clinical drugs used as positive controls. Based on their similarity in ED profiles, BFRs were classified into five different clusters. These findings support further investigation of the potential ED effects of these environmentally relevant BFRs in man and wildlife.
Glucocorticoids (GCs) are widely used as anti-inflammatory drugs. Our previous study demonstrated that several GCs such as cortisol and dexamethasone (Dex) were frequently detected in effluents ...collected from Japanese sewage treatment plants (STPs) in 2012. In this study, we used the GC-Responsive Chemical-Activated LUciferase gene eXpression (GR-CALUX) assay to elucidate GC receptor (GR) agonistic activities of ten pure synthetic GCs and selected STP effluents in Japan for assessment of the risks associated with the presence of GR agonists. The tested GCs demonstrated dose-dependent agonistic effects in the GR-CALUX assay and their EC50 values were calculated for estimation of relative potencies (REPs) compared to Dex. The GR agonistic potency was in the rank of: clobetasol propionate>clobetasone butyrate>betamethasone 17-valerate>difluprednate>betamethasone 17,21-dipropionate>Dex>betamethasone>6α-methylprednisolone>prednisolone>cortisol. The GR agonistic activity in STP effluents as measured in Dex-equivalent (Dex-EQ) activities ranged from <3.0–78ngL−1 (median: 29ngL−1, n=50). To evaluate the contribution of the target GCs, theoretical Dex-EQs were calculated by multiplying the concentrations of each GC by its respective REP. Our calculation of Dex-EQ contribution for individual GR agonists indicated that the well-known GCs cortisol and Dex should not be given priority for subsequent in vivo testing, monitoring and removal experiments, but rather the highly potent synthetic GCs clobetasol propionate and betamethasone 17-valerate (REP=28 and 3.1) as well as other unidentified compounds are important GR agonists in STP effluents in Japan.
•GR agonistic activity was detected in STP effluents from Japan for the first time.•Dex-EQ levels ranged from <3.0–78 ng L-1 in STP effluents.•Cortisol and Dex were not the major contributors to GR agonistic activity.•Other GCs and unidentified compounds might be more important GR agonists.
It is generally known that there are compounds present in the aquatic environment that can disturb endocrine processes, for example via interaction with the endogenous hormone receptors. Most ...research so far has focused on compounds that bind to the estrogen and/or androgen receptor, but ligands for other hormone receptors might also be present. In this study, a newly completed panel of human cell derived CALUX reporter gene bioassays was utilized to test water extracts for estrogen (ER), as well as androgen (AR), progesterone (PR), and glucocorticoid (GR) receptor mediated transactivation activity. Effluents from industry, hospital, and municipal sewage treatment plants, as well as tap water and different sources of surface water were tested. The CALUX reporter gene panel showed high sensitivity and specificity to known agonists, enabling discrimination between different receptor based endocrine responses present in the aquatic environment. Our results clearly showed the presence of agonistic activity on the ER, as well as on the AR, PR, and GR in the raw and wastewater and surface water extracts. However, no hormone receptor-mediated transactivation was detected in the drinking water or in the blank water. The levels of estrogenic activity were 0.2−0.5 ng E2-equiv/L for surface water and 0.4−1.0 ng E2-equiv/L for municipal effluents, which was consistent with previous studies. Surprisingly, the other hormonal activities were found to be present in similar or much higher levels. Most notably, glucocorticoid-like activity was detected in all samples, at surprisingly high levels ranging from 0.39−1.3 ng Dex-equiv/L in surface water and 11−243 ng Dex-equiv/L in effluents. When regarding the fact that dexamethasone in the GR CALUX bioassay is a factor 12 more potent than the natural hormone cortisol, results expressed as cortisol equivalents would range up to 2900 ng cortisol equiv/L. Further studies are needed to establish the identity of the active compounds and to understand the significance of the level of activities with regard to human and ecotoxicological risks.
Indoor dust is a sink for many kinds of pollutants, including flame retardants (FRs), plasticizers, and their contaminants and degradation products. These pollutants can be migrated to indoor dust ...from household items such as televisions and computers. To reveal high-priority end points of and contaminant candidates in indoor dust, using CALUX reporter gene assays based on human osteosarcoma (U2OS) cell lines, we evaluated and characterized the endocrine-disrupting potencies of crude extracts of indoor dust collected from Japan (n = 8), the United States (n = 21), Vietnam (n = 10), the Philippines (n = 17), and Indonesia (n = 10) and for 23 selected FRs. The CALUX reporter gene assays used were specific for compounds interacting with the human androgen receptor (AR), estrogen receptor α (ERα), progesterone receptor (PR), glucocorticoid receptor (GR), and peroxisome proliferator-activated receptor γ2 (PPARγ2). Indoor dust extracts were agonistic to ERα, GR, and PPARγ2 and antagonistic against AR, PR, GR, and PPARγ2. In comparison, a majority of FRs was agonistic to ERα and PPARγ2 only, and some FRs demonstrated receptor-specific antagonism against all tested nuclear receptors. Hierarchical clustering clearly indicated that agonism of ERα and antagonism of AR and PR were common, frequently detected end points for indoor dust and tested FRs. Given our previous results regarding the concentrations of FRs in indoor dust and in light of our current results, candidate contributors to these effects include not only internationally controlled brominated FRs but also alternatives such as some phosphorus-containing FRs. In the context of indoor pollution, high-frequency effects of FRs such as agonism of ERα and antagonism of AR and PR are candidate high-priority end points for further investigation.
Abstract
Recent evidence suggests that the interaction of polycyclic aromatic hydrocarbons (PAHs), present in some petroleum substances (PS), with particular nuclear-hormone-receptors and/or the ...dioxin (aryl hydrocarbon receptor AhR) receptor, may play a role in the prenatal developmental toxicity (PDT) induced by these substances. To address this hypothesis, we evaluated the possible endocrine and dioxin-like activity of the dimethylsulfoxide (DMSO)-extracts of 9 PS, varying in PAH content, and 2 gas-to-liquid (GTL) products, containing no PAHs but having similar other properties as PS, using a series of Chemical Activated LUciferase gene eXpression (CALUX) assays. The results show that the extracts of PS tested in this study possess various endocrine and dioxin-like activities and these in vitro potencies are associated with the quantity and type of PAHs they contain. All tested DMSO-extracts of PS show a strong AhR agonist activity and rather weak antiprogesterone, antiandrogen, and estrogenic activities. In the assays that evaluate thyroid-related and antiestrogen activity, only minor effects of specific extracts, particularly those with a substantial amount of 4–5 ring PAHs, ie, sample No. 34, 98, and 99, were observed. None of the GTL extracts interacted with the selected receptors. Of all assays, the AhR agonist activity correlates best (R2 = 0.80) with the in vitro PDT of the substances as quantified previously in the embryonic stem cell test, suggesting an important role of the AhR in mediating this effect. Hierarchic clustering of the combined CALUX data clustered the compounds in line with their chemical characteristics, suggesting a PS class-specific effects signature in the various CALUX assays, depending on the PAH profile. To conclude, our findings indicate a high potential for endocrine and dioxin-like activity of some PS extracts which correlates with their in vitro PDT and is driven by the PAHs present in these substances.
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•From 26 different countries, on four continents (Africa, America, Asia and Europe), >60% of the toys and consumer products analysed in this study by chemical (GC-HRMS) and ...effect-based analysis (DR CALUX) for polyhalogenated dioxins, showed higher levels than the proposed limit value for toxic waste (>1000 TEQ pg/g) (Basel Convention, 2019).•We measured high levels of PBDD/Fs in these black plastic parts by using DR CALUX (from 110 to 17,000 pg BEQ/g) and by GC-HRMS (levels up to 13,900 pg TEQ/g).•High TBBPA levels were measured by using TTR-TRβ CALUX (up to 410 µg/g) and by chemical analysis (up to 836 µg/g) indicating thyroid hormone disruption activities.•Effect-based CALUX bioassay for dioxin- and thyroid hormone-like activities picked-out all elevated toxic products showing that in vitro toxicity analysis of total PBDD/F and TBBPA is a promising and suitable strategy to cover complex mixtures of BFRs and to assess BFRs in consumer products in general.•This study add further evidence that current limits for both trace contamination and the definition of POPs waste set in the EU POPs Regulation (European Parliament and the Council of the European Union, 2019 and 2022) and for the total content of PBDEs are too weak (500 ppm) to protect human health. It will allow a large number of such contaminated products to enter the market.
Children and consumers are exposed to increasingly complex mixtures of known and as-yet-unknown toxic chemicals from toys and products. However traditional chemical analysis methods only evaluate a small number of chemicals at a time thereby restricting consumer awareness of the full range of potentially harmful chemicals in products.
We used high-throughput effect-based non-animal methods to investigate exposures to complex chemical mixtures of several kinds of brominated flame retardants (BFRs) for their dioxin- and thyroid hormone-like activities in various kinds of consumer products and toys from 26 different countries, on four continents (Africa, America, Asia and Europe) in combination with chemical analysis of various polybrominated flame retardants (BFRs) and their impurities (such as polyhalogenated PCDD/Fs and PBDD/Fs).
We found high levels of polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) in toys and now, for the first time, also in consumer products that are manufactured from black plastics containing certain brominated flame retardants (BFRs). The presence of PBDD/PBDFs as well as other BFRs in various black plastic materials from additional countries as well as additional kinds of consumer products as confirmed by effect-based in vitro reporter gene DR CALUX and TTR-TRβ CALUX assays as well as congener-specific chemical analysis. We compared total Toxicity Equivalent (TEQ) levels of PBDD/F-TEQs analysed by chemical analysis to by CALUX bioassay measured Biological equivalence (BEQ) concentrations (for further info see at ISO 23196, ISO, 2022). In the case of TBBPA, both chemical and TTR-TRβ CALUX analysis measure direct the amount of TBBPA. Finally, the daily ingestion of 2,3,7,8-TCDD equivalents from PBDD/Fs-contaminated plastic toys by child mouthing habits have been related to our earlier study (Budin et al., 2020).
Interaction of children with such contaminated plastics may significantly contribute to the daily uptake of dioxin- and thyroid hormone transport disrupting-like compounds. Effect-based bioassays for dioxin- and thyroid hormone-like activities are relevant to pick-out such complex mixtures of known and yet unknown (and therefore not regulated) substances for safer and more sustainable plastics. Low POPs Content Levels and other mechanisms set under the Basel and Stockholm Conventions are set far too high to prevent a significant flow of BFRs and PBDD/Fs into consumer products.
Occurrence of pharmaceuticals in aquatic ecosystems is related to sewage effluents. Due to the possible adverse effects on wildlife and humans, degradation and removal of pharmaceuticals and their ...metabolites during wastewater treatment is an increasingly important task.
The present study was part of a proof of concept study at a medium sized country hospital in western Germany that investigated efficiency of advanced treatment processes to remove toxic potencies from sewage. Specifically, the efficiency of treatment processes such as a membrane bioreactor (MBR) and ozonation to remove endocrine disruptive potentials was assessed. Estrogenic effects were characterized by use of two receptor-mediated in vitro transactivation assays, the Lyticase Yeast Estrogen Screen (LYES) and the Estrogen Receptor mediated Chemical Activated LUciferase gene eXpression (ER CALUX®). In addition, the H295R Steroidogenesis Assay (H295R) was utilized to detect potential disruption of steroidogenesis. Raw sewage contained measurable estrogen receptor (ER)-mediated potency as determined by use of the LYES (28.9 ± 8.6 ng/L, 0.33× concentration), which was reduced after treatment by MBR (2.3 ± 0.3 ng/L) and ozone (1.2 ± 0.4 ng/L). Results were confirmed by use of ER CALUX® which measured concentrations of estrogen equivalents (EEQs) of 0.2 ± 0.11 ng/L (MBR) and 0.01 ± 0.02 ng/L (ozonation). In contrast, treatment with ozone resulted in greater production of estradiol and aromatase activity at 3× and greater concentrations in H295R cells. It is hypothesized that this is partly due to formation of active oxidized products during ozonation. Substance-specific analyses demonstrated efficient removal of most of the measured compounds by ozonation. A comparison of the ER-mediated responses measured by use of the LYES and ER CALUX® with those from the chemical analysis using a mass-balance approach revealed estrone (E1) to be the main compound that caused the estrogenic effects. Overall, treatment of sewage by use of MBR successfully reduced estrogenicity of hospital effluents as well as substances that are able to alter sex steroid production. However, after ozonation, effluents should undergo further investigations regarding the formation of endocrine active metabolites. The results obtained as part of this study demonstrated applicability of in vitro assays for monitoring of endocrine-modulating potency of treated sewage.
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► Membrane bioreactor treatment significantly reduces the estrogenic burden of sewage. ► A combination of MBR and ozone almost completely reduces estrogenicity. ► Ozonation increased stimulation of endogenous estrogen production in H295R cells. ► Assessment with receptor-mediated and non-receptor-mediated assays is recommended.
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