Brita T.A. Muyssen

Uncertainties in the Environmental Risk Assessment of Metals

As life has evolved in the presence of metals, the assessment of the potential adverse effects of metals on ecosystems requires a different approach than those presently used for man-made organic substances. This article provides a brief review of applications and limitations of current techniques and presents, based on recent research results, suggestions for improving the scientific relevance and accuracy of environmental risk assessments of metals. The importance of the following factors responsible for major uncertainties in current environmental risk assessments of metals are discussed: factors affecting metal bioavailability and toxicity, the potential importance of deficiency effects (for essential metals), and field extrapolation of laboratory toxicity data. Possible (regulatory) consequences of inaccurately assessing the natural background concentrations of metals and acclimatization/adaptation potential of laboratory organisms and resident communities are illustrated using examples of recent research, hypothesis development, and a probabilistic environmental risk assessment.

Tolerance and acclimation to zinc of field-collected Daphnia magna populations

The zinc tolerance of two Daphnia magna populations collected at a zinc contaminated site was studied. One clone was isolated from each population in order to determine interclonal variation in zinc tolerance. 48hEC50-values, life table parameters, carapace lengths and cellular energy allocation (CEA) were used as test endpoints and compared with the results obtained with a standard laboratory clone. The natural clones were more tolerant to acute zinc toxicity (up to a factor of 4) and exhibited a higher reproduction rate (factor 2) and carapace length (factor 1.2). The optimal zinc concentrations for the natural clones ranged from 80 to 200 microg Zn/l. When cultured without zinc, the natural clones gradually lost their zinc tolerance. Therefore, the environmental relevance of using toxicity data obtained with organisms (natural, as well as laboratory clones) acclimated to culture media containing no or very small amounts of zinc can be questioned.

Zinc acclimation and its effect on the zinc tolerance of Raphidocelis subcapitata and Chlorella vulgaris in laboratory experiments.

The effect of zinc acclimation of Raphidocelis subcapitata (syn. Selenastrum capricornutum) and Chlorella vulgaris on their sensitivity towards this metal was examined in a series of laboratory experiments. These two commonly used algal species were acclimated to 65 microg Zn/l and changes in zinc tolerance were monitored using standard growth inhibition tests. The chemically defined ISO medium was used as a control culture medium. Both species demonstrated a maximum increase in zinc tolerance of a factor of 3 after 100 days of acclimation. Shifts in the shape of the concentration-response curve due to acclimation were observed for R. subcapitata. Compared to non-acclimated algae, acclimated R. subcapitata exhibited higher growth rates in all zinc treatments as well as in the controls. This suggests that the use of ISO-medium results in sub-optimal growth due to zinc deficiency. These effects could not be demonstrated for C. vulgaris. The zinc tolerance of both species decreased significantly one week after returning the acclimated algae to control (ISO) medium. 72hEC50 values based on growth rate were two to four times higher than those calculated using biomass measurements. Algal toxicity test results, particularly if used for metal risk assessments, must not be conducted using nutrient deficient media.

Importance of acclimation to environmentally relevant zinc concentrations on the sensitivity of Daphnia magna toward zinc

Daphnia magna was acclimated for six generations to an acclimation range of 0.02 to 74 microg/L of Zn2+. This range was determined by combining physicochemical water characteristics of European surface waters with total Zn concentrations in these waters in such a way that they resulted in minimal and maximal free (i.e., assumed bioavailable) Zn ion activities. No significant differences were found in acute Zn tolerance between the different acclimation concentrations: Average 48-h median effective concentration (EC50) values ranged from 608+/-94 to 713+/-249 microg/L of Zn2+. Also, no significant shifts in chronic tolerance were observed: Average 21-d EC50 (based on net reproductive rate) ranged from 91+/-20 to 124+/-22 microg/L of Zn2+. However, at test concentrations less than the 21-d EC50, acclimation significantly increased the reproductive capacity of the offspring produced. This indicates that metal acclimation is not necessarily accompanied by an increase in tolerance but also may manifest in other responses (e.g., reproduction rate). Organisms acclimated to a range from 6 to 22 microg/L of Zn2+ produced significantly more offspring than organisms acclimated to lower and higher Zn concentrations in test concentrations up to 50 microg/L of Zn2+. This range corresponds to a previously established optimal concentration range for D. magna. Bioconcentration factors indicated that Zn was actively regulated in the acclimation range tested.

Combined cadmium and temperature acclimation in Daphnia magna: physiological and sub-cellular effects.

Effects of temperature and Cd acclimation (>or=6 generations) on life history and tolerance responses to stress in three clones of Daphnia magna was examined using a 2x2 design (20 and 24 degrees C, 0 and 5 microg L(-1) Cd). Endpoints include acute Cd and heat tolerance, individual traits such as ingestion rates, growth and reproduction responses and physiological attributes such as acute Cd and heat tolerance, energy reserves, electron transport system activity, haemoglobin and oxidative stress enzymes. Cd (20 degrees C+Cd) did reduce reproduction, but acclimation to 24 degrees C+Cd did not decrease reproductive output additionally. For energy reserves, on which Cd and temperature acted similarly, no synergistic effect could be demonstrated. Generally, the effect of 24 degrees C+Cd was comparable to that of the 24 degrees C acclimation. Cd acclimation at 20 degrees C resulted in organisms, which were more tolerant to acute Cd and heat shock challenge, while the contrary was observed at 24 degrees C. A relationship between tolerance to Cd and heat shock and superoxide dismutase (SOD) activity was observed. Significant interclonal variation and genotypexenvironmental interactions in the measured traits evidenced that clones responded differently. As natural populations are invariably exposed to multiple stressors and genetic variability may change accordingly, it is essential to improve our knowledge on the effects of such scenarios in order to allow a correct incorporation in ecological risk assessment methodologies.

Tolerance and acclimation to zinc of Ceriodaphnia dubia.

Zinc is an essential metal for all living organisms. However, so far, little or no attention has been paid to the consequences of zinc deficiency or acclimation to this metal during culturing and testing on toxicity test results. In this study, the cladoceran Ceriodaphnia dubia was acclimated for 10 generations to four zinc concentrations ranging from 0 to 100 microg Zn/l and changes in zinc tolerance were monitored using acute (48 h) and chronic (9 days) assays. C. dubia deprived of zinc and acclimated to 13 microg Zn/l had a lower fitness in comparison with organisms acclimated to 50 and 100 microg Zn/l. In the two lowest versus the two highest acclimation concentrations the 9dEC50 values (on immobility) were 358-387 microg Zn/l versus 486-489 microg Zn/l; the mean number of young per female was 11-18 versus 25-32; and the time to first brood was 4.7-5.0 days versus 4.0-4.3 days. Moreover, the coefficient of variation of all parameters tested was highest in the two lowest acclimation concentrations. The results indicate that culturing test animals in media lacking trace metals such as zinc could give rise to animals that are unnaturally sensitive to those same metals daring toxicity tests.

The micro-evolutionary potential of Daphnia magna population exposed to temperature and cadmium stress.

This study examines micro-evolutionary aspects of a natural Daphnia magna population exposed to Cd. To this end, a set of hypotheses related to micro-evolutionary responses and to how these are influenced by temperature and Cd stress, were tested. Life-table experiments were conducted with 14 D. magna clones collected from an unpolluted lake following a 2x2 design with Cd concentration and temperature as the factors (control vs. 5 microg/L cadmium, 20 vs. 24 degrees C). Several fitness traits were monitored during 21 days. Our results demonstrate (1) that chemicals can have effects on key population genetic characteristics such as genetic variation and between-trait correlations and (2) that these effects may differ depending on temperature. Their findings also suggests that further research is needed to understand the importance of combined chemical-global warming stress for micro-evolutionary responses of organisms. These aspects are currently not accounted for in any regulatory environmental risk assessment procedure.

Integration of molecular with higher-level effects of dietary zinc exposure in Daphnia magna.

We exposed Daphnia magna for 21 days to dietary Zn, incorporated in a diet of the green alga Pseudokirchneriella subcapitata at 720 microg Zn/g dry wt and compared its response to D. magna fed with a control diet (116 microg Zn/g dry wt). Exposure to dietary Zn resulted in an increased body burden of D. magna (93.7 microg/g dry wt vs. 61.3 microg/g dry wt in the control) but did not affect survival, growth, or feeding rate. Only reproduction was significantly reduced from the 2nd brood onwards. Gene expression analysis, using microarray analysis and RT-PCR, showed that dietary Zn exposure resulted in the differential expression of several genes involved in molting-associated processes (i.e., chitin binding, cuticle metabolism), especially after 6 days of exposures (but not after 13 or 21 days of exposure). Monitoring of time to molt and intermolt-period confirmed this molting effect at the organism level in the first week of exposure. The data suggest a possible link between Zn-induced effects on molting-related processes and reproductive inhibition, but this link is only obvious for effects on the 2nd brood size and not for later broods. Reproductive inhibition in later broods may also be explained by a disturbed mitochondrial function, but more research is clearly needed to give a more definitive integrated explanation of the observed effects at the molecular and organism level.

Calcium accumulation and regulation in Daphnia magna: Links with feeding, growth and reproduction

Calcium is involved in a wide variety of biological processes and has an important structural role in crustaceans. The present study aimed at exploring the possible link between Ca body concentrations and the ingestion rate and the role of soft tissue vs. total tissue Ca accumulation in Daphnia magna. D. magna was cultured for 21 days at different water Ca concentrations ranging from 3.4 to 32.5 mg/L. Every week Ca body concentrations (soft and total tissues), ingestion rate, growth, survival and reproduction were measured. Daily, algal food that was not deficient in Ca was supplied. Ca in the soft tissues represented 8 to 26% of the total Ca body concentrations. The ratio Ca in soft tissue/Ca in total tissue was generally not influenced by the Ca exposure concentration but decreased with time, i.e., age (from an average of 0.24 at day 7 to 0.09 at day 21). During week 1, a 54% decrease in Ca body concentrations was observed in daphnids exposed in medium with 3.4 mg/L Ca compared to those exposed to 32.5 mg/L. The concurrent decrease in ingestion rate was 14%. No significant differences among Ca treatments were observed during week 2 for ingestion rate and week 3 for calcium body concentrations. Also, no effects on growth and reproduction were observed, although these were expected at the lowest Ca concentration tested. It is hypothesised that Ca absorption from food in combination with an increased ingestion rate is used to maintain Ca homeostasis under Ca limiting conditions.

Relevance of generic and site‐specific species sensitivity distributions in the current risk assessment procedures for copper and zinc

Species sensitivity distributions (SSD) were constructed using acute toxicity data of various cladoceran species collected in five different aquatic systems. The aim of this research was to study the relative acute cladoceran community sensitivity in different aquatic systems. Current risk assessment procedures are based upon hypothetical communities and do not take into account variation in species composition and tolerance between aquatic communities. Two metals, copper and zinc, were used as model toxicants. To establish comparative sensitivity, a standard medium (International Organization for Standardization [ISO]) was used. The generic SSD (log-normal distribution) based on toxicity data obtained in this standard medium for all species (collected at all sites) resulted in a hazardous concentrations that protects 95% of the species occurring in a (hypothetical) ecosystem (i.e., hazardous concentration protecting 95% of the species of the hypothetical ecosystem [HC5]) of 6.7 microg Cu L(-1) (90% confidence limits: 4.2-10.8) and 559 microg Zn L(-1) (375-843). This generic SSD was not significantly different from the site-specific SSDs (i.e., constructed with species only occurring at a specific site). Mean community sensitivity (the geometric mean of 48-h 50% effective concentration [EC50] values of species within a community) among sites varied within a factor of 2 (between 17.3 and 23.6 microg Cu L(-1) for Cu and between 973 and 1,808 microg Zn L(-1) for Zn), and HC5s varied within a factor of 4 for copper (between 4.5 and 17.3 microg Cu L(-1)) and 7 for zinc (between 194 and 1,341 microg Zn L(-1)). For copper, the HC50 of our generic SSD was significantly lower than the one based on literature toxicity data of cladoceran species (which were recalculated to the hardness of our standard medium). In contrast, no significant differences were observed between the generic SSD and the literature-based SSD for zinc. It is suggested that the community sensitivity of different cladoceran populations is similar among aquatic systems and is not dependent on the species composition.