Nico M. van Straalen

Ecotoxicological evaluation of soil quality criteria

To implement the Soil Protection Act of 1986, the Dutch Ministry of Housing, Physical Planning, and Environment has recently proposed a list of soil quality reference values. These values are, as yet, insufficiently based on ecotoxicological evidence. In this paper, a three-step procedure of risk assessment for soil contaminants is proposed. Arguing from experimental results concerning no observed effect concentrations for a set of selected soil organisms, the method aims at protecting a certain fraction of soil life, taking factors such as soil organic matter and clay content into account. When applied to cadmium, a concentration protecting 95% of soil invertebrates is estimated as 0.16 micrograms/g for a standard soil. The value of 0.8 micrograms/g, as proposed by the Dutch authorities, may, given the present variation and uncertainty of toxicity data, protect about 85% of the soil invertebrate fauna. It is concluded that even low levels of cadmium in soil may endanger the functioning of some sensitive soil animal species.

A Review of the Effects of Multiple Stressors on Aquatic Organisms and Analysis of Uncertainty Factors for Use in Risk Assessment

Risk assessment procedures use toxicity tests in which organisms are subjected to chemicals under otherwise constant and favorable experimental conditions. Because variable and suboptimal environmental conditions are common aspects of natural ecosystems, the hazard of underestimation of risk arises. Therefore, an uncertainty factor is used in the extrapolation of results of standard toxicity tests to field situations. The choice for these uncertainty factors is based on little ecological evidence. This review discusses studies on the toxicity of various chemicals to aquatic organisms, modified by temperature, nutritional state and salinity, excluding papers on changes in bioavailability of compounds. Collected data were analyzed quantitatively to evaluate the validity of toxicity data obtained from standard toxicity tests in the laboratory under field conditions. Generally, organisms living under conditions close to their environmental tolerance limits appeared to be more vulnerable to additional chemical stress. Usually, increasing temperature and decreasing food or nutrient level raised toxicity. The influence of salinity was less clear; metal toxicity increased with decreasing salinity, toxicity of organophosphate insecticides increased with higher salinity, while for other chemicals no clear relationship between toxicity and salinity was observed. The interactions can be explained by several physical and physiological processes, acting on factors such as bioavailability, toxicokinetics, and sensitivity of organisms. Quantitative analysis of data indicated that an uncertainty factor for the laboratory to field extrapolation should be smaller than one for an ecosystem in a temperate region, while a factor greater than one would be appropriate for systems nearby discharge points of cooling water. The factor should be greater than one when varying nutritional state is concerned, but smaller than one with respect to salinity. Dependent on the effect parameter used, the differences in toxicity between laboratory and relevant field situations ranged from a factor of 2.6 to 130 and 1.7 to 15 for the two temperature conditions and 1.2 to 10 for nutritional state. A salinity increase from freshwater to marine water decreased toxicity by a factor of 2.1. However, as less extreme salinity changes are more relevant under field conditions, the change in toxicity is probably much smaller. To obtain uncertainty factors that sufficiently protect natural systems without being overprotective, additional research is required.

Heavy-metal adaptation in terrestrial invertebrates: A review of occurrence, genetics, physiology and ecological consequences

Abstract 1. The occurrence of genetic adaptation to heavy metals in natural populations of terrestrial invertebrates is evaluated from literature data. Five criteria for adaptation evidence are applied, with concepts from ecotoxicology, ecology, life-history theory and quantitative genetics. 2. There is strong evidence for the occurrence of adaptation in natural populations of the isopod Porcellio scaber (Isopoda), the springtails Isotoma notabilis , Onychiurus armatus and Orchesella cincta (Collembola), the blowfly Lucilia cuprina and the fruit fly Drosophila melanogaster (Diptera). Adaptation to metal-containing pesticides has been demonstrated in ticks (Acarina). Population divergence indicates acclimation or adaptation in many other species. 3. Metal adaptation has been achieved within a few generations under laboratory conditions in some species; adapted populations occur at field sites that have been polluted for decades, or longer. 4. Genetic variation for tolerance and life-history characteristics, allowing for adaptation, was quantified in a reference population of Orchesella cincta . Tolerance and life-history patterns in exposed field populations matched predictions from genetic variation. 5. Adaptation involves modification and intensification of existing physiological mechanisms for metal assimilation, excretion, immobilization or compartmentalized storage. There are indications ofinter-population divergence in metal-binding proteins in a snail. In the fruit fly Drosophila melanogaster metal adaptation is achieved by duplication of the metallothionein gene. 6. An altered life-history is often part of the complex adaptation syndrome. Metal-adapted invertebrates have a shorter life-cycle and a higher reproductive effort. 7. Possible consequences of adaptation, consisting of costs of tolerance determined by genetic correlations, and probably of reduced genetic variation for tolerance and other features, are discussed. Reduced genetic variation is suggested by results for the springtail Orchesella cincta . 8. The distinction between “costs of tolerance” on the one hand and linkage disequilibrium or direct selection for altered life-history patterns on the other hand is discussed. 9. Species with high sensitivity (i.e. a low NOEC), that do not have populations maintaining sufficient genetic variation to evolve tolerance or modified life-history characteristics, or that have costly tolerance mechanisms, or both, are most at risk for extinction at sites with increasing metal pollution. 10. Metal adaptation in terrestrial invertebrates appears to be of degree rather than of kind: indications for a specific metal-fauna, equivalent to metal-vegetation, are lacking.

Genetic variation in toxicant-stressed populations: an evaluation of the "genetic erosion" hypothesis.

The question whether environmental pollution affects genetic diversity in natural populations remains unanswered to date despite the fact that genetic variation is one of the three pillars of biodiversity recognized in the Rio convention of 1993. The loss of genetic diversity in populations subjected to anthropogenic stress can be designated as “genetic erosion” and may be considered as a factor of concern in risk assessment of toxic chemicals. Theoretically there are four different ways in which toxicants can affect genetic variation: (i) by increasing mutation rates, (ii) by directional selection on tolerant genotypes, (iii) by causing bottleneck events, and (iv) by altering migration. This paper reviews studies that have documented genetic change in animal populations exposed to environmental pollution. In these studies, genetic variation is measured in a variety of ways: heritability of quantitative characters, heterozygosity of allozyme loci, haplotype diversity in mitochondrial DNA, and variability in RAPD fingerprints. Studies on cadmium tolerance of Collembola living in metal-contaminated soil suggest that strong directional selection pressure may decrease genetic variability of traits immediately linked to tolerance. Allozyme studies in fish have documented a similar decrease of genetic variation in populations living in strongly acidified waters. A correlation between RAPD-PCR-based genetic similarity and site contamination has been documented in crayfish. Overall, there is significant support for the genetic erosion hypothesis, but the issue cannot be considered settled. In most studies insufficient attention is given to factors such as population size, bottlenecks and mutation, which may influence genetic variability in addition to the toxicant selection regime. At the moment, there does not seem to be a sound scientific basis for incorporating genetic diversity measurements into risk assessment, despite the variety of easily applicable molecular techniques available. It is often not known what kind of variation is measured by these techniques (neutral or selectable) and how the markers are inherited. Given the importance of the issue, as stressed by the Rio Convention, a concentrated research effort is necessary to better define the question and find a general approach to evaluate its importance in ecological risk assessment.

Heavy metal content of forest litter arthropods as related to body-size and trophic level

Abstract Lead, zinc and cadmium concentrations were measured in thirteen species of small forest floor arthropods, inhabiting an area polluted by zinc factory emissions in the Netherlands. The species covered three orders of magnitude in body-sizes (3 μg to 6 mg), and a variety of feeding habits. Analysis of covariance was used to correct for weight effects on body burden. Metal analysis on an individual basis revealed large differences between species, even with similar feeding habits. The data are discussed in the light of two conflicting ecotoxicological theories which state that residues of chemicals either depend on body-size, or on the trophic level. The results show that, although body-size considerations may be relevant with respect to lead, both theories are inadequate with respect to zinc and cadmium. The concentrations of these metals seem to be connected to the physiological equipment of species, rather than to body-size or trophic level.

Adaptation to soil pollution by cadmium excretion in natural populations of Orchesella cincta (L.) (Collembola).

Population differentiation in Orchesella cincta (L.) (Collembola) populations, from various heavy metal contaminated sites, was studied by comparing cadmium excretion efficiency in first generation (F1) laboratory individuals. Animals from sites with high metal concentrations in the litter and with a long history of contamination showed significantly higher excretion efficiencies than animals from low pollution, or reference sites. Differences found in the F1 laboratory animals suggest evidence for genetic differences between the populations.Beneficial and detrimental effects of cadmium excretion were studied in relation to body growth and cadmium concentrations. In chronically exposed animals from an unpolluted site, no physiological acclimation was observed. Excretion efficiency was negatively correlated with body concentrations of cadmium. No detrimental effects were found.Whole-body equilibrium concentrations of cadmium and lead were similar in F1 animals from the reference site and polluted sites. Significant differences in excretion efficiencies imply that the distribution of toxic metals over body compartments differs, tolerant populations having a higher proportion deposited in the gut. Body concentrations of zinc were consistently higher in animals from the polluted site, during both cadmium and zinc exposure. No detrimental effects of increased cadmium excretion on body concentrations of zinc were observed. Population comparisons of cadmium excretion efficiency data and growth reduction in F1 laboratory animals showed that both parameters were inversely related. Cadmium and lead contamination were not the sole factors determining tolerance differentiation.

Genetic variation and covariation for characteristics associated with cadmium tolerance in natural populations of the springtail Orchesella cincta (L.)

Heavy metals can be strong and stable directional selective agents for metal‐exposed populations. Genetic variation for the metal‐tolerance characteristic “cadmium excretion efficiency” was studied in populations of the collembolan Orchesella cincta from a reference‐ and a metal‐contaminated forest soil. Previously it has been shown that “excretion efficiency” influences tolerance through midgut‐mediated immobilization and excretion of toxic metal ions, and that an increased mean excretion efficiency is present in animals inhabiting metal‐contaminated litter. In the present research, offspring‐parent regressions showed that additive genetic variation for cadmium excretion efficiency was present in the population from the reference site. The heritability estimate was 0.33. In the natural population exposed to heavy metals from an industrial source, additive genetic variation was not significantly different from zero. Differences in the heritability between the reference and the exposed population were not significant. Genetic variation for cadmium excretion efficiency allows for a response to selection in the reference population. Such a response has probably occurred in the metal‐exposed population. Half‐sib analysis with animals from the reference population was used to estimate genetic variation and maternal effects for excretion efficiency, relative growth rate and molting frequency, and to determine genetic correlations between these characteristics. Additive genetic variation was demonstrated for all three characteristics, heritability estimates were 0.48, 0.75 and 0.46, respectively. Maternal effects were low for excretion efficiency and molting frequency, but may be present for relative growth rate. Phenotypic and genetic correlations among these characteristics were positive. The environmental correlation between relative growth rate and molting frequency was positive, others were negative. Direct selection for any of the characteristics, or genetic correlations between tolerance characteristics and growth characteristics, or both may have caused the responses previously observed in field populations.

Population growth of Daphnia magna under multiple stress conditions: joint effects of temperature, food, and cadmium.

Aquatic organisms in the field often are exposed to combinations of stress factors of various origins. Little is known of the interaction between different types of stressors; hence, the predictability of their joint effects is low. Therefore, the present study analyzed the joint effects of temperature, food, and cadmium on the population growth rate of the water flea Daphnia magna. The results revealed that temperature, food, and cadmium, as well as their interactions, were important factors that influenced life-history parameters and, as a consequence, the population growth rate of D. magna. In general, population growth rate increased at high temperature and food level but decreased when cadmium was present. The positive effect of temperature on population growth rate was smallest at limiting food levels. Negative effects of cadmium on the growth rate were enhanced at elevated temperatures, whereas high food levels protected the daphnids from adverse effects of cadmium. To avoid over- or underestimation regarding the toxicity of substances to field populations, results of standard toxicity tests should be applied in a location-specific way.

Molecular mechanisms of heavy metal tolerance and evolution in invertebrates.

Following the genomics revolution, our knowledge of the molecular mechanisms underlying defenses against stress has been greatly expanded. Under strong selective pressure many animals may evolve an enhanced stress tolerance. This can be achieved by altering the structure of proteins (through mutations in the coding regions of genes) or by altering the amount of protein (through changes in transcriptional regulation). The latter type of evolution can be achieved by substitutions in the promoter of the gene of interest (cis‐regulatory change) or by altering the structure or amount of transcriptional regulator proteins (trans‐regulatory change). The metallothionein system is one of the best studied stress response systems in the context of heavy metals. Metallothionein expression is assumed to be regulated by metal transcription factor 1 (MTF‐1); however, up to now the involvement of MTF‐1 has only been proven for some vertebrates and Drosophila. Data on invertebrates such as nematodes and earthworms suggest that other mechanisms of metallothionein induction may be present. A detailed study of Cd tolerance was done for a species of soil‐living springtail, Orchesella cincta. The metallothionein gene of this species is overexpressed in metal‐exposed field populations. Analysis of the metallothionein promoter has demonstrated extensive polymorphisms that have a functional significance, as shown in bioreporter assays. In a study comparing 20 different populations, the frequency of a high‐expresser promoter allele was positively correlated with the concentration of metals in soil, especially Cd. The springtail study shows that cis‐regulatory change of genes involved in the cellular stress response may contribute to evolution of metal tolerance.

Biological significance of metals partitioned to subcellular fractions within earthworms (Aporrectodea caliginosa)

Metal ions in excess of metabolic requirements are potentially toxic and must be removed from the vicinity of important biological molecules to protect organisms from adverse effects. Correspondingly, metals are sequestrated in various forms, defining the accumulation pattern and the magnitude of steady-state levels reached. To investigate the subcellular fractions over which Ca, Mg, Fe, Cu, Zn, Cd, Pb, Ni, and As are distributed, earthworms (Aporrectodea caliginosa) collected from the field were analyzed by isolating metal-rich granules and tissue fragments from intracellular microsomal and cytosolic fractions (i.e., heat-stable proteins and heat-denatured proteins). The fractions showed metal-specific binding capacity. Cadmium was mainly retrieved from the protein fractions. Copper was equally distributed over the protein fraction and the fraction comprising tissue fragments, cell membranes, and intact cells. Zinc, Ca, Mg, and As were mainly found in this fraction as well. Lead, Fe, and Ni were mainly isolated from the granular fraction. To study accumulation kinetics in the different fractions, three experiments were conducted in which earthworms were exposed to metal-spiked soil and a soil contaminated by anthropogenic inputs and, indigenous earthworms were exposed to field soils. Although kinetics showed variation, linear uptake and steady-state types of accumulation patterns could be understood according to subcellular compartmentalization. For risk assessment purposes, subcellular distribution of metals might allow for a more precise estimate of effects than total body burden. Identification of subcellular partitioning appears useful in determining the biological significance of steady-state levels reached in animals.