Effects of environmental conditions and catch processing on survival probability of plaice discards in the North Sea pulse trawl fishery.

Undersized European plaice dominate the discarded fraction of the catch of the beam trawl fisheries for sole in the Southern North Sea. Effects of environmental conditions at sea and of the use of a water-filled hopper on the survival of undersized European plaice discarded by pulse trawl fisheries were explored. During trips with commercial pulse-trawlers catches were discharged in either water-filled hoppers or conventional dry hoppers. For both hoppers, undersized plaice were sampled from the sorting belt. After assessment of vitality status, sampled fish were housed in dedicated survival monitoring tanks on board. Upon return in the harbour fish were transferred to the laboratory to monitor their survival for up to 18 days post-catch. Conditions at sea, such as wave height and water temperature, as prevailing during these trips were recorded or obtained from public data sources. The overall estimate for the survival probability for plaice discarded by pulse trawl fisheries is 12% (95% CI: 8% - 18%). Both water temperature and vitality status had strong effects on survival probabilities of discarded plaice. Increasing water temperature increased mortality. The vitality of the fish could be moderately increased by using a water-filled hopper to collect the fish on deck, but we found no significant direct effect of hopper type on plaice discard survival. It seems that to increase discards survival, fish need to be landed on deck in much better condition by a reduction of the impact of capture and hauling processes on fish condition.

Correction to: Lack of adverse effects in subchronic and chronic toxicity/carcinogenicity studies on the glyphosate-resistant genetically modified maize NK603 in Wistar Han RCC rats.

In the original publication, the starting point in time for the three feeding trials.

Statistical models discriminating between complex samples measured with microfluidic receptor-cell arrays.

Data analysis for flow-based in-vitro receptomics array, like a tongue-on-a-chip, is complicated by the relatively large variability within and between arrays, transfected DNA types, spots, and cells within spots. Simply averaging responses of spots of the same type would lead to high variances and low statistical power. This paper presents an approach based on linear mixed models, allowing a quantitative and robust comparison of complex samples and indicating which receptors are responsible for any differences. These models are easily extended to take into account additional effects such as the build-up of cell stress and to combine data from replicated experiments. The increased analytical power this brings to receptomics research is discussed.

Equivalence analysis to support environmental safety assessment: Using nontarget organism count data from field trials with cisgenically modified potato.

This paper considers the statistical analysis of entomological count data from field experiments with genetically modified (GM) plants. Such trials are carried out to assess environmental safety. Potential effects on nontarget organisms (NTOs), as indicators of biodiversity, are investigated. The European Food Safety Authority (EFSA) gives broad guidance on the environmental risk assessment (ERA) of GM plants. Field experiments must contain suitable comparator crops as a benchmark for the assessment of designated endpoints. In this paper, a detailed protocol is proposed to perform data analysis for the purpose of assessing environmental safety. The protocol includes the specification of a list of endpoints and their hierarchical relations, the specification of intended levels of data analysis, and the specification of provisional limits of concern to decide on the need for further investigation. The protocol emphasizes a graphical representation of estimates and confidence intervals for the ratio of mean abundances for the GM plant and its comparator crop. Interpretation relies mainly on equivalence testing in which confidence intervals are compared with the limits of concern. The proposed methodology is illustrated with entomological count data resulting from multiyear, multilocation field trials. A cisgenically modified potato line (with enhanced resistance to late blight disease) was compared to the original conventional potato variety in the Netherlands and Ireland in two successive years (2013, 2014). It is shown that the protocol encompasses alternative schemes for safety assessment resulting from different research questions and/or expert choices. Graphical displays of equivalence testing at several hierarchical levels and their interpretation are presented for one of these schemes. The proposed approaches should be of help in the ERA of GM or other novel plants.

Lack of adverse effects in subchronic and chronic toxicity/carcinogenicity studies on the glyphosate-resistant genetically modified maize NK603 in Wistar Han RCC rats.

In 2012, a controversial study on the long-term toxicity of a Roundup herbicide and the glyphosate-tolerant genetically modified (GM) maize NK603 was published. The EC-funded G-TwYST research consortium tested the potential subchronic and chronic toxicity as well as the carcinogenicity of the glyphosate-resistant genetically modified maize NK603 by performing two 90-day feeding trials, one with GM maize inclusion rates of 11 and 33% and one with inclusion rates of up to 50%, as well as a 2-year feeding trial with inclusion rates of 11 and 33% in male and female Wistar Han RCC rats by taking into account OECD Guidelines for the testing of chemicals and EFSA recommendations on the safety testing of whole-food/feed in laboratory animals. In all three trials, the NK603 maize, untreated and treated once with Roundup during its cultivation, and the conventional counterpart were tested. Differences between each test group and the control group were evaluated. Equivalence was assessed by comparing the observed difference to differences between non-GM reference groups in previous studies. In case of significant differences, whether the effects were dose-related and/or accompanied by changes in related parameters including histopathological findings was evaluated. It is concluded that no adverse effects related to the feeding of the NK603 maize cultivated with or without Roundup for up to 2 years were observed. Based on the outcome of the subchronic and combined chronic toxicity/carcinogenicity studies, recommendations on the scientific justification and added value of long-term feeding trials in the GM plant risk assessment process are presented.

An Integrated System for the Automated Recording and Analysis of Insect Behavior in T-maze Arrays.

Host-plant resistance to insects like thrips and aphids is a complex trait that is difficult to phenotype quickly and reliably. Here, we introduce novel hardware and software to facilitate insect choice assays and automate the acquisition and analysis of movement tracks. The hardware consists of an array of individual T-mazes allowing simultaneous release of up to 90 insect individuals from their individual cage below each T-maze with choice of two leaf disks under a video camera. Insect movement tracks are acquired with computer vision software (EthoVision) and analyzed with EthoAnalysis, a novel software package that allows for automated reporting of highly detailed behavior parameters and statistical analysis. To validate the benefits of the system we contrasted two Arabidopsis accessions that were previously analyzed for differential resistance to western flower thrips. Results of two trials with 40 T-mazes are reported and we show how we arrived at optimized settings for the different filters and statistics. The statistics are reported in terms of frequency, duration, distance and speed of behavior events, both as sum totals and event averages, and both for the total trial period and in time bins of 1 h. Also included are higher level analyses with subcategories like short-medium-long events and slow-medium-fast events. The time bins showed how some behavior elements are more descriptive of differences between the genotypes during the first hours, whereas others are constant or become more relevant at the end of an 8 h recording. The three overarching behavior categories, i.e., choice, movement, and halting, were automatically corrected for the percentage of time thrips were detected and 24 out of 38 statistics of behavior parameters differed by a factor 2-6 between the accessions. The analysis resulted in much larger contrasts in behavior traits than reported previously. Compared to leaf damage assays on whole plants or detached leaves that take a week or more to complete, results were obtained in 8 h, with more detail, fewer individuals and higher significance. The potential value of the new integrated system, named EntoLab, for discovery of genetic traits in plants and insects by high throughput screening of large populations is discussed.

Equivalence limit scaled differences for untargeted safety assessments: Comparative analyses to guard against unintended effects on the environment or human health of genetically modified maize.

Safety assessments guard against unintended effects for human health and the environment. When new products are compared with accepted reference products by broad arrays of measurements, statistical analyses are usually summarised by significance tests or confidence intervals per endpoint. The traditional approach is to test for statistical significance of differences. However, absence or presence of significant differences is not a statement about safety. Equivalence limits are essential for safety assessment. We propose graphs to present the results of equivalence tests over the array of endpoints. It is argued that plots of the equivalence limit scaled difference (ELSD) are preferable over plots of the standardised effect size (SES) used previously for similar assessments. The ELSD method can be used either with externally specified equivalence limits or with equivalence limits estimated from (historical) data. The method is illustrated with two examples: first, environmental safety of MON810 Bt maize was assessed using field trial count data of arthropods; second, human safety of herbicide tolerant NK603 maize was assessed using haematological, biochemical and organ weight data from a 90-day rat feeding study. All assessed endpoints were classified in EFSA equivalence categories I or II, implying full equivalence or equivalence more likely than not.

Equivalence testing using existing reference data: An example with genetically modified and conventional crops in animal feeding studies.

An equivalence testing method is described to assess the safety of regulated products using relevant data obtained in historical studies with assumedly safe reference products. The method is illustrated using data from a series of animal feeding studies with genetically modified and reference maize varieties. Several criteria for quantifying equivalence are discussed, and study-corrected distribution-wise equivalence is selected as being appropriate for the example case study. An equivalence test is proposed based on a high probability of declaring equivalence in a simplified situation, where there is no between-group variation, where the historical and current studies have the same residual variance, and where the current study is assumed to have a sample size as set by a regulator. The method makes use of generalized fiducial inference methods to integrate uncertainties from both the historical and the current data.

The MCRA model for probabilistic single-compound and cumulative risk assessment of pesticides.

Pesticide risk assessment is hampered by worst-case assumptions leading to overly pessimistic assessments. On the other hand, cumulative health effects of similar pesticides are often not taken into account. This paper describes models and a web-based software system developed in the European research project ACROPOLIS. The models are appropriate for both acute and chronic exposure assessments of single compounds and of multiple compounds in cumulative assessment groups. The software system MCRA (Monte Carlo Risk Assessment) is available for stakeholders in pesticide risk assessment at mcra.rivm.nl. We describe the MCRA implementation of the methods as advised in the 2012 EFSA Guidance on probabilistic modelling, as well as more refined methods developed in the ACROPOLIS project. The emphasis is on cumulative assessments. Two approaches, sample-based and compound-based, are contrasted. It is shown that additional data on agricultural use of pesticides may give more realistic risk assessments. Examples are given of model and software validation of acute and chronic assessments, using both simulated data and comparisons against the previous release of MCRA and against the standard software DEEM-FCID used by the Environmental Protection Agency in the USA. It is shown that the EFSA Guidance pessimistic model may not always give an appropriate modelling of exposure.

Can plants grow on Mars and the moon: a growth experiment on Mars and moon soil simulants.

When humans will settle on the moon or Mars they will have to eat there. Food may be flown in. An alternative could be to cultivate plants at the site itself, preferably in native soils. We report on the first large-scale controlled experiment to investigate the possibility of growing plants in Mars and moon soil simulants. The results show that plants are able to germinate and grow on both Martian and moon soil simulant for a period of 50 days without any addition of nutrients. Growth and flowering on Mars regolith simulant was much better than on moon regolith simulant and even slightly better than on our control nutrient poor river soil. Reflexed stonecrop (a wild plant); the crops tomato, wheat, and cress; and the green manure species field mustard performed particularly well. The latter three flowered, and cress and field mustard also produced seeds. Our results show that in principle it is possible to grow crops and other plant species in Martian and Lunar soil simulants. However, many questions remain about the simulants' water carrying capacity and other physical characteristics and also whether the simulants are representative of the real soils.

Why some plant species are rare.

Biodiversity, including plant species diversity, is threatened worldwide as a result of anthropogenic pressures such as an increase of pollutants and climate change. Rare species in particular are on the verge of becoming extinct. It is still unclear as to why some plant species are rare and others are not. Are they rare due to: intrinsic reasons, dispersal capacity, the effects of management or abiotic circumstances? Habitat preference of rare plant species may play an important role in determining why some species are rare. Based on an extensive data set of soil parameters we investigated if rarity is due to a narrow habitat preference for abiotic soil parameters. For 23 different abiotic soil parameters, of which the most influential were groundwater-table, soil-pH and nutrient-contents, we estimated species responses for common and rare species. Based on the responses per species we calculated the range of occurrence, the range between the 5 and 95 percentile of the response curve giving the habitat preference. Subsequently, we calculated the average response range for common and rare species. In addition, we designed a new graphic in order to provide a better means for presentation of the results. The habitat preferences of rare species for abiotic soil conditions are significantly narrower than for common species. Twenty of the twenty-three abiotic parameters showed on average significantly narrower habitat preferences for rare species than for common species; none of the abiotic parameters showed on average a narrower habitat preference for common species. The results have major implications for the conservation of rare plant species; accordingly management and nature development should be focussed on the maintenance and creation of a broad range of environmental conditions, so that the requirements of rare species are met. The conservation of (abiotic) gradients within ecosystems is particularly important for preserving rare species.

A statistical simulation model for field testing of non-target organisms in environmental risk assessment of genetically modified plants.

Genetic modification of plants may result in unintended effects causing potentially adverse effects on the environment. A comparative safety assessment is therefore required by authorities, such as the European Food Safety Authority, in which the genetically modified plant is compared with its conventional counterpart. Part of the environmental risk assessment is a comparative field experiment in which the effect on non-target organisms is compared. Statistical analysis of such trials come in two flavors: difference testing and equivalence testing. It is important to know the statistical properties of these, for example, the power to detect environmental change of a given magnitude, before the start of an experiment. Such prospective power analysis can best be studied by means of a statistical simulation model. This paper describes a general framework for simulating data typically encountered in environmental risk assessment of genetically modified plants. The simulation model, available as Supplementary Material, can be used to generate count data having different statistical distributions possibly with excess-zeros. In addition the model employs completely randomized or randomized block experiments, can be used to simulate single or multiple trials across environments, enables genotype by environment interaction by adding random variety effects, and finally includes repeated measures in time following a constant, linear or quadratic pattern in time possibly with some form of autocorrelation. The model also allows to add a set of reference varieties to the GM plants and its comparator to assess the natural variation which can then be used to set limits of concern for equivalence testing. The different count distributions are described in some detail and some examples of how to use the simulation model to study various aspects, including a prospective power analysis, are provided.

Olivine weathering in soil, and its effects on growth and nutrient uptake in Ryegrass (Lolium perenne L.): a pot experiment.

Mineral carbonation of basic silicate minerals regulates atmospheric CO(2) on geological time scales by locking up carbon. Mining and spreading onto the earth's surface of fast-weathering silicates, such as olivine, has been proposed to speed up this natural CO(2) sequestration ('enhanced weathering'). While agriculture may offer an existing infrastructure, weathering rate and impacts on soil and plant are largely unknown. Our objectives were to assess weathering of olivine in soil, and its effects on plant growth and nutrient uptake. In a pot experiment with perennial ryegrass (Lolium perenne L.), weathering during 32 weeks was inferred from bioavailability of magnesium (Mg) in soil and plant. Olivine doses were equivalent to 1630 (OLIV1), 8150, 40700 and 204000 (OLIV4) kg ha(-1). Alternatively, the soluble Mg salt kieserite was applied for reference. Olivine increased plant growth (+15.6%) and plant K concentration (+16.5%) in OLIV4. At all doses, olivine increased bioavailability of Mg and Ni in soil, as well as uptake of Mg, Si and Ni in plants. Olivine suppressed Ca uptake. Weathering estimated from a Mg balance was equivalent to 240 kg ha(-1) (14.8% of dose, OLIV1) to 2240 kg ha(-1) (1.1%, OLIV4). This corresponds to gross CO(2) sequestration of 290 to 2690 kg ha(-1) (29 10(3) to 269 10(3) kg km(-2).) Alternatively, weathering estimated from similarity with kieserite treatments ranged from 13% to 58% for OLIV1. The Olsen model for olivine carbonation predicted 4.0% to 9.0% weathering for our case, independent of olivine dose. Our % values observed at high doses were smaller than this, suggesting negative feedbacks in soil. Yet, weathering appears fast enough to support the 'enhanced weathering' concept. In agriculture, olivine doses must remain within limits to avoid imbalances in plant nutrition, notably at low Ca availability; and to avoid Ni accumulation in soil and crop.

Comparison of bioassessment results and costs between preserved and unpreserved macroinvertebrate samples from streams.

The choice to use or not use a preservative before sorting macroinvertebrate samples (i.e., dead specimens vs. living specimens) is based on studies not solely focused on the effects of preservation. Using identical sample processing protocols, we compared preserved and unpreserved samples for the following parameters: (1) the number of taxa and individuals for each major macroinvertebrate group, (2) ecological quality classes calculated with a multimetric index developed for the assessment of small Dutch lowland streams, and (3) costs of sample processing. We collected macroinvertebrate samples from three lowland streams in the Netherlands. At each site, we collected six replicate samples, of which three samples were preserved and three were not. Significantly different numbers of Ephemeroptera individuals and Hydracarina taxa and individuals were collected from preserved samples compared to unpreserved samples. In assessments based on these individual metrics, standardization of sample processing will be required. In streams with Ephemeroptera, the preservation of samples is necessary to optimize the number of Ephemeroptera individuals collected. In streams that contain Hydracarina, the preservation of samples will result in an underestimation of the number of Hydracarina taxa and individuals present. In only one instance there was a difference in ecological quality between preserved and unpreserved samples, indicating that assessing small Dutch lowland streams does not require standardization of sample preservation as part of the sample processing protocol. We detected no significant differences in sample processing costs between preserved and unpreserved samples.

Field effects of pollutants in dynamic environments. A case study on earthworm populations in river floodplains contaminated with heavy metals.

In industrialized countries river floodplains can be strongly polluted with heavy metals. Published studies on effects of heavy metal pollution on soil invertebrates in floodplains, however, are inconclusive. This is unexpected since studies in other less dynamic environments reported clear effects at even lower levels of pollution. Flooding induces extra variation in invertebrate biomass and abundance which may reduce the probability to detect heavy metal effects. In this paper we combine reported data from studies on river floodplains in The Netherlands and Belgium and statistically analyze the effect of heavy metals on species composition, biomass, density and individual weight of earthworms. Interaction effects of heavy metal stress and flooding are also considered. The results suggest clear effects of zinc and copper on all variables and interaction of heavy metals and flooding for individual weight.