Biological effect and chemical monitoring of Watch List substances in European surface waters: Steroidal estrogens and diclofenac - Effect-based methods for monitoring frameworks.

Three steroidal estrogens, 17α-ethinylestradiol (EE2), 17ß-estradiol (E2), estrone (E1), and the non-steroidal anti-inflammatory drug (NSAID), diclofenac have been included in the first Watch List of the Water Framework Directive (WFD, EU Directive 2000/60/EC, EU Implementing Decision 2015/495). This triggered the need for more EU-wide surface water monitoring data on these micropollutants, before they can be considered for inclusion in the list of priority substances regularly monitored in aquatic ecosystems. The revision of the priority substance list of the WFD offers the opportunity to incorporate more holistic bioanalytical approaches, such as effect-based monitoring, alongside single substance chemical monitoring. Effect-based methods (EBMs) are able to measure total biological activities (e.g., estrogenic activity or cyxlooxygenase [COX]-inhibition) of specific group of substances (such as estrogens and NSAIDs) in the aquatic environment at low concentrations (pg/L). This makes them potential tools for a cost-effective and ecotoxicologically comprehensive water quality assessment. In parallel, the use of such methods could build a bridge from chemical status assessments towards ecological status assessments by adressing mixture effects for relevant modes of action. Our study aimed to assess the suitability of implementing EBMs in the WFD, by conducting a large-scale sampling and analysis campaign of more than 70 surface waters across Europe. This resulted in the generation of high-quality chemical and effect-based monitoring data for the selected Watch List substances. Overall, water samples contained low estrogenicity (0.01-1.3 ng E2-Equivalent/L) and a range of COX-inhibition activity similar to previously reported levels (12-1600 ng Diclofenac-Equivalent/L). Comparison between effect-based and conventional analytical chemical methods showed that the chemical analytical approach for steroidal estrogens resulted in more (76%) non-quantifiable data, i.e., concentrations were below detection limits, compared to the EBMs (28%). These results demonstrate the excellent and sensitive screening capability of EBMs.

Anti-androgenic compounds in breast milk and cryptorchidism among Norwegian boys in the HUMIS birth cohort.

The prevalence of cryptorchidism has increased over the past decades, yet its origins remain poorly understood. Testis descent is dependent on androgens and likely affected by endocrine disrupting compounds (EDCs), targeting the androgen receptor (AR). We investigated the association between anti-androgenic activity, not derived from natural hormones, in maternal breast milk and impaired testis descent among boys. We performed a case-control study based on 199 breast milk samples from 94 mothers of cryptorchid boys and 105 random non-cryptorchid boys participating in the Norwegian HUMIS (Human Milk Study) cohort. For each participant, apolar, and polar fractions were extracted, and combined to reconstitute a mixture. Anti-androgenic activity was measured in all three fractions using the human cell-based in vitro anti-AR CALUX® assay and expressed in µg of flutamide equivalent, a well-known antiandrogen. Results from fraction analyses were compared among boys with cryptorchidism and controls using multiple logistic regression, controlling for appropriate confounders identified using a directed acyclic graph. Children's daily exposure to anti-androgenic EDCs through breastfeeding was estimated to 78 µg flutamide eq./kg of body weigh/day. The activity was higher in the polar fraction (1.48 ± 1.37 µg flutamide eq./g of milk) mainly representing non-persistent chemicals, in contrast to other fractions. However, the activity in the polar extracts was decreased when in mixtures with the apolar fraction, indicating synergistic interactions. No significant difference in the activity was observed according to cryptorchid status for polar, apolar or mixed breast milk fractions. The study showed anti-androgenic activity in nearly all human milk samples, and at levels higher than the advisory threshold. However, no significant association was observed between cryptorchidism and antiandrogenic activity measured in either polar, apolar, or mixture fractions derived from breast milk.

Characterization of 2,3,7,8-tetrachlorodibenzo-p-dioxin biodegradation by extracellular lignin-modifying enzymes from ligninolytic fungus.

Ligninolytic fungi secrete extracellular lignin-modifying enzymes (LME) that degrade plant polymers for fungal nutrition but that are, because of their broad substrate specificity, also applicable for the degradation of many hazardous pollutants. Laccase is one of the most well characterized LME and is involved in the removal and degradation of recalcitrant aromatic compounds with or without the assistance of laccase-mediators. The Ligninolytic fungus Rigidoporus sp. FMD21 can degrade 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) with a half-life of 6.2 days. Using Rigidoporus sp. FMD21 crude extracellular enzyme extract (ExE) that mainly consisted of laccase, 77.4% of 2,3,7,8-TCDD was degraded within 36 days. The degradation rate did not depend on the 2,3,7,8-TCDD concentration in the tested range between 0.005 and 0.5 pgTEQ/µL. 2,3,7,8-TCDD was analysed by DR-CALUX® bioassay and the degradation was confirmed by GC-HRMS. In this study, we found evidence for cleavage of the diaryl ether bond in the 2,3,7,8-TCDD molecule and here we propose a new degradation mechanism in which 3,4-dichlorophenol is the main metabolite of 2,3,7,8-TCDD degradation by FMD21's ExE. Six laccase-mediators were tested. Three of them 1-hydroxybenzotriazole (HBT), syringaldehyde (Syr) and violuric acid (Vio) showed an equipotent added effect on 2,3,7,8-TCDD degradation by ExE, however only in case of Vio a level of significance was reached. The others showed no effect or negatively impacted degradation. In conclusion, we have shown that Rigidoporus sp. FMD21 produces extracellular enzymes, mainly laccases that apparently are able to degrade the highly recalcitrant and most toxic 2,3,7,8-congener of TCDD via diaryl bond cleavage into 3,4-dichlorophenol.

Effect-directed analysis and chemical identification of agonists of peroxisome proliferator-activated receptors in white button mushroom.

Obesity has a serious effect on human health. It relates to metabolic syndrome, including the associated disorders such as type 2 diabetes, heart disease, stroke and hyperemia. The peroxisome proliferator-activated receptors (PPARs) are important receptors to control fat metabolism in the human body. Because of the safety concerns of synthetic drugs targeting PPARs, ligands from natural sources have drawn interest. Earlier, we have found high PPAR activities in extracts from Agaricus bisporus (white button mushroom, WBM). WBM contains a wide range of candidate compounds which could be agonists of PPARs. To identify which compounds are responsible for PPAR activation by WBM extracts, we used fractionation coupled to effect-directed analysis with reporter gene assays specific for all three PPARs for purification and LC/MS-TOF and NMR for compound identification in purified active fractions. Surprisingly, we identified the relatively common dietary fatty acid, linoleic acid, as the main ligand of PPARs in WBM. Possibly, the relatively low levels of linoleic acid in WBM are sufficient and instrumental in inducing its anti-obesogenic effects, avoiding high energy intake and negative health effects associated with high levels of linoleic acid consumption. However, it could not be excluded that a minor relatively potent compound contributes towards PPAR activation, while the anti-obesity effects of WBM may be further enhanced by receptor expression modulating compounds or compounds with completely PPAR unrelated modes of action.

Antagonistic activity towards the androgen receptor independent from natural sex hormones in human milk samples from the Norwegian HUMIS cohort.

In this paper, we investigated the possible presence of endocrine disrupting chemicals (EDCs) based on measuring the total estrogenic and androgenic activity in human milk samples. We used specific bioassays for analysis of the endocrine activity of estrogens and estrogen-like EDCs and androgens and androgen-like EDCs and developed a separation method to evaluate the contribution from natural hormones in comparison to that of EDCs to total endocrine activities. We extracted ten random samples originating from the Norwegian HUMIS biobank of human milk and analyzed their agonistic or antagonistic activity using the ERα- and AR CALUX® bioassays. The study showed antagonistic activity towards the androgen receptor in 8 out of 10 of the assessed human milk samples, while 2 out of 10 samples showed agonistic activity for the ERα. Further investigations demonstrated anti-androgenic activity in the polar fraction of 9 out of 10 samples while no apolar extracts scored positive. The culprit chemicals causing the measured antagonistic activity in AR CALUX was investigated through liquid chromatography fractionation coupled to bioanalysis and non-target screening involving UHPLC-Q-TOF-MS/MS, using a pooled polar extract. The analysis revealed that the measured anti-androgenic biological activity could not be explained by the presence of endogenous hormones nor their metabolites. We have demonstrated that human milk of Norwegian mothers contained anti-androgenic activity which is most likely associated with the presence of anthropogenic polar EDCs without direct interferences from natural sex hormones. These findings warrant a larger scale investigation into endocrine biological activity in human milk, as well as exploring the chemical sources of the activity and their potential effects on health of the developing infant.

An evidence-based approach to organization evaluation and change in human service organizations evaluation and program planning.

The work described in this article focuses primarily on how human service organizations can use an evidence-based, self-assessment approach to organization evaluation to facilitate continuous quality improvement and organization change. Real-life examples are presented, strengths and challenges discussed, and future conceptual and measurement issues identified.

Pesticide residues in sediments and aquatic species in Lake Nokoué and Cotonou Lagoon in the Republic of Bénin.

Lake Nokoué and Cotonou Lagoon are the most important and most productive continental freshwaters in Bénin, with an estimated fish production of over 2 tonnes per hectare in Lake Nokoué. Organochlorine pesticides are used in agriculture and to repel tsetse flies, malaria mosquitoes and other diseases raised. Sediment, fish, shrimp and oyster species were collected in Lake Nokoué and Cotonou Lagoon for pesticide residues analysis. The main pesticides identified in sediment were pp'-DDT and its metabolites pp-DDE and pp'-DDD, with residue levels between the detection limit and 24.4 µg/kg dry weight. Fish species commonly consumed such as Elops lacerta, Podamasys jubelini, Gobbienellus occidentalis, Ethmalosa fimbriata, Mugil cephalus and Hemichromis fasciatus were contaminated with residues of seven to nine pesticides, including pp-DDE, op'-DDD, pp'-DDD, op'-DDT, pp'-DDT, α-endosulfan, aldrin, dieldrin and γ-hexachlorocyclohexane. The levels ranged from detection limit to 289 ng/g lipid. The same pesticides were also detected in other aquatic species, such as shrimp and oysters. A summed risk assessment, comparing pesticide intake levels through fish consumption with tolerable daily intake levels proposed by the World Health Organization, showed in all cases a low risk for human health.

Perfluoroalkyl substances in polar bear mother-cub pairs: a comparative study based on plasma levels from 1998 and 2008.

Perfluoroalkyl substances (PFASs) are protein-binding blood-accumulating contaminants that may have detrimental toxicological effects on the early phases of mammalian development. To enable an evaluation of the potential health risks of PFAS exposure for polar bears (Ursus maritimus), an exposure assessment was made by examining plasma levels of PFASs in polar bear mothers in relation to their suckling cubs-of-the-year (~4 months old). Samples were collected at Svalbard in 1998 and 2008, and we investigated the between-year differences in levels of PFASs. Seven perfluorinated carboxylic acids (∑7PFCAs: PFHpA, PFOA, PFNA, PFDA, PFUnDA, PFDoDA, and PFTrDA) and two perfluorinated sulfonic acids (∑2PFSAs: PFHxS and PFOS) were detected in the majority of the mothers and cubs from both years. In mothers and cubs, most PFCAs were detected in higher concentrations in 2008 than in 1998. On the contrary, levels of PFOS were lower in 2008 than in 1998, while levels of PFHxS did not differ between the two sampling years. PFOS was the dominating compound in mothers and cubs both in 1998 and in 2008. Concentration of PFHpA did not differ between mothers and cubs, while concentrations of PFOA, PFNA, PFDA, PFUnDA, PFDoDA, PFTrDA, PFHxS, and PFOS were higher in mothers than in their cubs. Except from PFHpA, all compounds correlated significantly between mothers and their cubs. The mean cub to mother ratios ranged from 0.15 for PFNA to 1.69 for PFHpA. On average (mean±standard error of mean), the levels of ∑7PFCAs and ∑2PFSAs in cubs were 0.24±0.01 and 0.22±0.01 times the levels in their mothers, respectively. Although maternal transfer appears to be a substantial source of exposure for the cubs, the low cub to mother ratios indicate that maternal transfer of PFASs in polar bears is relatively low in comparison with hydrophobic contaminants (e.g. PCBs). Because the level of several PFASs in mothers and cubs from both sampling years exceeded the levels associated with health effects in humans, our findings raise concern on the potential health effects of PFASs in polar bears from Svalbard. Effort should be made to examine the potential health effects of PFASs in polar bears.

Estrogenic and dioxin-like compounds in sediment from Zierikzee harbour identified with CALUX assay-directed fractionation combined with one and two dimensional gas chromatography analyses.

The identity of compounds responsible for estrogenic and dioxin-like activities in sediment from the harbour of the small town Zierikzee in Zeeland, The Netherlands, was investigated using a bioassay directed fractionation approach with the in vitro estrogen and dioxin responsive reporter gene assays ER- and DR-CALUX. For identification of compounds exhibiting activity in the bioassays, either one or two-dimensional GC in combination with quadrupole (MSD), ion trap (ITD) or time-of-flight mass spectrometric detection (ToF-MS) was used, depending on the biological and chemical characteristics and the complexity of the fractions. The natural estrogenic hormone 17-beta-estradiol and its metabolite estrone were identified with GC-ITD as the main contributors to the estrogenic activity. After successive rounds of fractionation, the dioxin-like activity could be explained by the presence of various polycyclic aromatic hydrocarbons identified with GC-MSD and two-dimensional comprehensive GC x GC-ToF-MS. Some estrogenic activity of a relatively non-polar nature remained unidentified.

Organochlorine and organophosphorous pesticide residues in the Ouémé River catchment in the Republic of Bénin.

The Ouémé River is one of the most important rivers in the Republic of Bénin. It is 510 km long and its catchment drains 75% of the country. In this study, organochlorine and organophosphorous pesticide residues were measured in more than 35 sediment samples collected on nine locations along the Ouémé River from upstream to downstream. Except for one location, Tanéka-Koko, all areas were contaminated by more than 20 pesticides. Organochlorine pesticides identified in sediment samples included pp'-DDE, op'-DDD, pp'-DDD, op'-DDT, pp'-DDT, alpha-endosulfan, beta-endosulfan, endosulfan sulphate, alpha-HCH, beta-HCH, lindane, aldrin, dieldrin, endrin, telodrin, isodrin, cis- and trans-heptachlorepoxide, hexachlorbutadiene, hexachlorobenzene and octachlorostyrene. The organophosphorous pesticide chlorpyrifos, used in a new formulation to protect cotton, was also identified. In some areas, the concentrations of organochlorine pesticides in the sediment of the Ouémé River exceeded environmental quality standards and are reason for concern.

Contamination of fish by organochlorine pesticide residues in the Ouémé River catchment in the Republic of Bénin.

In the Republic of Bénin, aquatic ecosystems are subject to poisoning risks due to the inappropriate use of pesticides, such as washing of empty bottles in rivers and using pesticides to catch fish. In some areas, cotton fields are located near riverbanks, increasing the probability of pesticide emission to the river. To assess contamination levels in the Ouémé River catchment area, different fish species were collected from different geographical areas along the river. DDT, its metabolites and isomers were the most frequently identified pesticides in fish flesh, alpha-endosulfan, beta-endosulfan, dieldrin, telodrin, lindane and octachlorostyrene were also detected. Concentrations of pesticide residues in fish ranged from 0 to 1364 ng/g lipid. A preliminary risk assessment indicated that the daily intake of chlorinated pesticides by people consuming fish from the Ouémé River still is rather low and does not present an immediate risk.