The OECD Validation Program of the H295R Steroidogenesis Assay for the Identification of In Vitro Inhibitors and Inducers of Testosterone and Estradiol Production. Phase 2: Inter-Laboratory Pre-Validation Studies (8 pp)

Background, Goals and Scope In response to concerns that have been raised about chemical substances that may alter the function of endocrine systems and result in adverse effects on human health, an OECD initiative was undertaken to develop and validate in vitro and in vivo assays to identify chemicals that may interfere with endocrine systems of vertebrates. Here we report on studies that were conducted to develop and standardize a cell-based screening assay using the H295R cell line to prioritize chemicals that may act on steroidogenic processes in humans and wildlife. These studies are currently ongoing as part of the ‘Special Activity on the Testing and Assessment of Endocrine Disruptors’ within the OECD Test Guidelines Program to review, develop, standardize, and validate a number of in vitro and in vivo toxicological assays for testing and assessment of chemicals concerning their potential to interact with the endocrine system of vertebrates. Study Design Six laboratories from five countries participated in the pre-validation studies. Each laboratory tested the effects of three model chemicals on the production of testosterone (T) and estradiol (E2) using the H295R Steroidogenesis Assay. Chemicals tested were well described inducers or inhibitors of steroidogenic pathways (forskolin, prochloraz and fadrozole). All experiments were conducted in 24 well plates following standard protocols. Six different doses per compound were analyzed in triplicate per plate. A quality control (QC) plate was run in conjunction with the chemical exposure plate to account for inter-assay variation. Each chemical exposure was conducted two or three times. Results All laboratories successfully detected increases and/or decreases in hormone production by H295R cells after exposure to the different model compounds and there was good agreement in the pattern of response for all groups. Forskolin increased both T and E2 while fadrozole and prochloraz decreased production of both hormones. All chemicals affected hormone production in a dose-dependent manner with the exception of fadrozole which caused maximum inhibition of E2 at the two least concentrations tested. Some inter-laboratory differences were noted in the alteration of hormone production measured in chemically exposed cells. However, with the exception of the production of T measured at one laboratory in cells exposed to forskolin, the EC₅₀s calculated were comparable (coefficients of variation 34–49%) for all hormones. Discussion and Perspectives The results indicated that the H295R Steroidogenesis Assay protocol was robust, transferable and reproducible among all laboratories. However, in several instances that were primarily related to one laboratory there were unexplained minor uncertainties related to the inter-laboratory hormone production variation. Based on the findings from this Phase 2 prevalidation study, the H295R Steroidogenesis Assay protocol is currently being refined. The next phase of the OECD validation program will test the refined protocol among the same group of laboratories using an extended set of chemicals (∼30) that will include positive and negative chemical controls as well as a broad spectrum of different potential inducers and inhibitors of steroidogenic pathways. Submission Editor: Dr. Carsten Brühl (bruehl@uni-landau.de)     

Response of zooxanthellae in symbiosis with the Mediterranean corals Cladocora caespitosa and Oculina patagonica to elevated temperatures

Scleractinian symbiotic corals living in the Ligurian Sea (NW Mediterranean Sea) have experienced warm summers during the last decade, with temperatures rapidly increasing, within a few days, to 3–4°C above the mean value of 24°C. The effect of elevated temperatures on the photosynthetic efficiency of zooxanthellae in symbiosis with temperate corals has not been well investigated. In this study, the corals, Cladocora caespitosa and Oculina patagonica were collected in the Ligurian Sea (44°N, 9°E), maintained during 2 weeks at the mean summer temperature of 24°C and then exposed during 48 h to temperatures of 24 (control), 27, 29 and 32°C. Chlorophyll (chl) fluorescence parameters [F _v/F _m, electron transport rate (ETR), non-photochemical quenching (NPQ)] were measured using pulse amplitude modulated (PAM) fluorimetry before, during the thermal increase, and after 1 and 7 days of recovery (corals maintained at 24°C). Zooxanthellae showed a broad tolerance to temperature increase, since their density remained unchanged and there was no significant reduction in their maximum quantum yield (F _v/F _m) or ETR up to 29°C. This temperature corresponded to a 5°C increase compared to the mean summer temperature (24°C) in the Ligurian Sea. At 32°C, there was a significant decrease in chl contents for both corals. This decrease was due to a reduction in the chl/zooxanthellae content. For C. caespitosa, there was also a decrease in ETR_max, not associated with a change in F _v/F _m or in the non-photochemical quenching (NPQ); for O. patagonica, both ETR_max and F _v/F _m significantly decreased, and NPQ_max showed a significant increase. Damages to the photosystem II appeared to be reversible in both corals, since F _v/F _m values returned to normal after 1 day at 24°C. Zooxanthellae in symbiosis with the Mediterranean corals investigated can therefore be considered as resistant to short-term increases in temperature, even well above the maximum temperatures experienced by these corals in summer.     

Ecological research for integrated coastal zone management: Introduction

Two Special Features on integrated coastal zone management, especially along the Mediterranean Sea, result from a MeDCOAST conference held in Hammamet (Tunisia). 20–25 October 2001. In this first Special Feature some papers are presented on ecological and biological research related to integrated coastal zone management. All over Europe coastal environments are threatened by human activities such as urbanization, industrial development, fisheries, aquaculture, recreation and tourism. Research has to be developed that can be applied to similar case studies in different countries. Methods of monitoring are necessary and baseline data have to be available in order to judge the significance of changes in the abjotic and biotic environment. Examples of studies included in this Special Feature are mainly from southern banks of the Mediterranean Sea but there is also an example from Estonia and one from the Canary Islands.     

Airborne observations of vegetation and implications for biogenic emission characterization

Measuring hydrocarbons from aircraft represents one way to infer biogenic emissions at the surface. The focus of this paper is to show that complementary remote sensing information can be provided by optical measurements of a vegetation index, which is readily measured with high temporal coverage using reflectance data. We examine the similarities between the vegetation index and in situ measurements of the chemicals isoprene, methacrolein, and alpha-pinene to estimate whether the temporal behavior of the in situ measurements of these chemicals could be better understood by the addition of the vegetation index. Data were compared for flights conducted around Houston in August and September 2000. The three independent sets of chemical measurements examined correspond reasonably well with the vegetation index curves for the majority of flight days. While low values of the vegetation index always correspond to low values of the in situ chemical measurements, high values of the index correspond to both high and low values of the chemical measurements. In this sense it represents an upper limit when compared with in situ data (assuming the calibration constant is adequately chosen). This result suggests that while the vegetation index cannot represent a purely predictive quantity for the in situ measurements, it represents a complementary measurement that can be useful in understanding comparisons of various in situ observations, particularly when these observations occur with relatively low temporal frequency. In situ isoprene measurements and the vegetation index were also compared to an isoprene emission inventory to provide additional insight on broad issues relating to the use of vegetation indices in emission database development.     

Improvement of the Bag-Mediated Filtration System for Sampling Wastewater and Wastewater-Impacted Waters

Environmental surveillance of poliovirus (PV) plays an important role in the global program for eradication of wild PV. The bag-mediated filtration system (BMFS) was first developed in 2014 and enhances PV surveillance when compared to the two-phase grab method currently recommended by the World Health Organization (WHO). In this study, the BMFS design was improved and tested for its usability in wastewater and wastewater-impacted surface waters in Nairobi, Kenya. Modifications made to the BMFS included the size, color, and shape of the collection bags, the filter housing used, and the device used to elute the samples from the filters. The modified BMFS concentrated 3–10 L down to 10 mL, which resulted in an effective volume assayed (900–3000 mL) that was 6–20 times greater than the effective volume assayed for samples processed by the WHO algorithm (150 mL). The system developed allows for sampling and in-field virus concentration, followed by transportation of the filter for further analysis with simpler logistics than the current methods. This may ultimately reduce the likelihood of false-negative samples by increasing the effective volume assayed compared to samples processed by the WHO algorithm, making the BMFS a valuable sampling system for wastewater and wastewater-impacted surface waters.     

Characteristics and predictors of recidivist drink-drivers

Objective: The current study compared characteristics of nonrecidivist versus recidivist drink-drivers and of recidivists in their first versus second appearance at Back on Track (BOT), the remedial measures program for impaired drivers in Ontario, Canada.

Methods: Information from 59,134 convicted drivers who participated in BOT between 2000 and 2010 was examined to identify drivers who completed the program a second time following reconviction.

Results: A total of 586 recidivists were identified. Compared to nonrecidivist drivers, recidivists at first attendance were more likely to be male and had higher scores on measures of alcohol dependence and adverse legal consequences of substance use. Compared to nonrecidivist drivers, recidivists at second attendance were significantly older, had a higher income, were more likely to be retired, and were less likely to be employed part-time. They had fewer legal problems. Recidivists reported fewer drinking days and fewer drinks per occasion but greater use of benzodiazepines than nonrecidivists and had higher scores on a measure of future risk of alcohol- and drug-related problems. Comparison of recidivists' characteristics at first versus second attendance confirmed many of these findings, with second-time recidivists reporting fewer drinks per drinking day and greater use of benzodiazepines and having higher scores on a measure of future substance use problems than first-time recidivists.

Conclusions: Results suggest that identification of drivers at increased risk of recidivism may be possible at first program attendance by examining indicators of increased alcohol-related problems. In addition, recidivists appear to show a greater readiness to change at second attendance. Implications for remedial program development and recommendations for future research are discussed.     

Adaptation of plant functional group composition to management changes in calcareous grassland

It is the aim of plant functional research to generate general rules of species assembly and species reactions based on plant functional traits. Here we determined plant functional groups that are important with regard to land use types (grazing, mowing, mulching, burning, abandonment) and looked for mechanisms leading to changes in species composition after management changes in calcareous grasslands.To filter out the set of traits which best described the variation in plant composition along the studied land use gradient and to identify functional groups we chose an iterative three-table ordination method. We included traits related to persistence as well as traits describing the germination niche of a plant. To gain insight into underlying mechanisms which led to differing importance of these plant functional groups at different management regimes we investigated shifts in dominance of the identified functional groups at eight management regimes using long-term vegetation data of a site which was grazed before the onset of different management regimes in 1974. Ongoing unidirectional changes in cover of the four determined functional groups at most of the treatments indicated that even after almost 30 years new equilibria of functional group composition were still not reached.The study showed that other management treatments but grazing and mowing led to changes in functional group composition, which were mainly driven by increasing dominance of a highly competitive species group.     

Reviewing the relevance of dioxin and PCB sources for food from animal origin and the need for their inventory,control and management

Background

In the past, cases of PCDD/F and PCB contamination exceeding limits in food from animal origin (eggs, meat or milk) were mainly caused by industrially produced feed. But in the last decade, exceedances of EU limit values were discovered more frequently for PCDD/Fs or dioxin-like(dl)-PCBs from free range chicken, sheep, and beef, often in the absence of any known contamination source.

Results

The German Environment Agency initiated a project to elucidate the entry of PCBs and PCDD/Fs in food related to environmental contamination. This paper summarizes the most important findings. Food products from farm animals sensitive to dioxin/PCB exposure—suckling calves and laying hens housed outdoor—can exceed EU maximum levels at soil concentrations that have previously been considered as safe. Maximum permitted levels can already be exceeded in beef/veal when soil is contaminated around 5 ng PCB-TEQ/kg dry matter (dm). For eggs/broiler, this can occur at a concentration of PCDD/Fs in soil below 5 ng PCDD/F–PCB-TEQ/kg dm. Egg consumers—especially young children—can easily exceed health-based guidance values (TDI). The soil–chicken egg exposure pathway is probably the most sensitive route for human exposure to both dl-PCBs and PCDD/Fs from soil and needs to be considered for soil guidelines. The study also found that calves from suckler cow herds are most prone to the impacts of dl-PCB contamination due to the excretion/accumulation via milk. PCB (and PCDD/F) intake for free-range cattle stems from feed and soil. Daily dl-PCB intake for suckler cow herds must in average be less than 2 ng PCB-TEQ/day. This translates to a maximum concentration in grass of 0.2 ng PCB-TEQ/kg dm which is less than 1/6 of the current EU maximum permitted level. This review compiles sources for PCDD/Fs and PCBs relevant to environmental contamination in respect to food safety. It also includes considerations on assessment of emerging POPs.

Conclusions

The major sources of PCDD/F and dl-PCB contamination of food of animal origin in Germany are (1) soils contaminated from past PCB and PCDD/F releases; (2) PCBs emitted from buildings and constructions; (3) PCBs present at farms. Impacted areas need to be assessed with respect to potential contamination of food-producing animals. Livestock management techniques can reduce exposure to PCDD/Fs and PCBs. Further research and regulatory action are needed to overcome gaps. Control and reduction measures are recommended for emission sources and new listed and emerging POPs to ensure food safety.

     

Deleterious effects of a nonPST bioactive compound(s) from <Emphasis Type="Italic">Alexandrium tamarense</Emphasis> on bivalve hemocytes

The known negative effects of shellfish toxin-producing dinoflagellates on feeding, burrowing and survival of some bivalve mollusks has prompted questions concerning whether they might also impair the internal defense system of affected bivalves and make them more susceptible to disease agents. The primary components of the cellular defense system are hemocytes. Many toxic dinoflagellates are too large to be ingested whole by hemocytes and would most likely be exposed to intracellular toxins only after the algae are consumed, broken down, and the water-soluble toxins, released. Therefore, we conducted a series of experiments in which hemocytes from two suspension-feeding bivalves—the Manila clam, Ruditapes philippinarum, and the softshell clam, Mya arenaria—were exposed in vitro to filtered extracts of one highly toxic paralytic shellfish toxin (PST)-producing and one nonPST-producing strain of Alexandrium tamarense (isolates PR18b, 76 ± 6 STXeq cell⁻¹ and CCMP115, with undetectable PST, respectively). We measured adherence and phagocytosis, two hemocyte attributes known to be inhibited by bacterial pathogens and other stressors. We found no measurable effect of a cell-free extract from a highly concentrated suspension of the PST-producing strain on hemocytes of either bivalve species. Instead, extract from the nonPST-producing strain had a consistent negative effect on both clams, resulting in significantly lower adherence and phagocytosis compared to strain PR18b and filtered seawater controls. The bioactive compound produced by strain CCMP115, which has yet to be characterized, may be similar to the PST-independent allelopathic compounds described for Alexandrium spp., which act on other plankters. These compounds and those produced by other harmful algae are known to cause immobilization, cellular deformation and lysis of co-occurring target organisms. Thus, nonPST producing Alexandrium spp., which do not cause paralysis and burrowing incapacitation of clams, may still produce a compound(s) that has negative effects not only on hemocytes, but on other molluscan cell types and their functions, as well.     

Developing a circumpolar programme for the monitoring of Arctic terrestrial biodiversity

The Arctic is undergoing biological and environmental changes, and a coordinated effort to monitor is critical to detect these changes. The Circumpolar Biodiversity Monitoring Programme (CBMP) of the Arctic Council biodiversity working group, Conservation of Arctic Flora and Fauna (CAFF), has developed pan-Arctic biodiversity monitoring plans that aims to improve the ability to detect and report on long-term changes. Whilst introducing this special issue, this paper also presents the making of the terrestrial monitoring plan and discusses how the plan follows the steps required for an adaptive and ecosystem-based monitoring programme. In this article, we discuss how data on key findings can be used to inform circumpolar and global assessments, including the State of the Arctic Terrestrial Biodiversity Report, which will be the first terrestrial assessment made by the CBMP. Key findings, advice for future monitoring and lessons learned will be used in planning next steps of pan-Arctic coordinated monitoring.