Whole sediment toxicity tests for metal risk assessments: on the importance of equilibration and test design to increase ecological relevance.

Current laboratory-based approaches for predicting metal toxicity in sediments exhibit a number of limitations. The most important are (1) a lack of sufficient equilibration resulting in unrealistically low pH values or unnaturally high porewater metal concentrations and (2) an inadequate test design regarding the metal concentrations selected for spiking. The present study illustrates that by explicitly accounting for these limitations, one obtains reliable and environmentally realistic toxicity data, thus advancing the metal risk assessments of sediments. To this end, a toxicity test design with natural sediments was developed in which the administered metal concentrations were selected to comprise a range of the difference between the molar concentration of simultaneously extracted metals and acid volatile sulfides (SEM-AVS) closely surrounding zero. In addition, the test design presented includes a 35- or 40-d equilibration period with overlying water renewal during which conductivity, pH, and metal concentrations in the overlying water are monitored. This allows toxicity testing to start after equilibrium for these parameters has been reached. This test design was applied to Ephoron virgo (Olivier, 1791), Gammarus pulex (Linnaeus, 1758), and Lumbriculus variegatus (Mueller, 1774) exposed to Zn and Pb. These tests indicated that the general concept of absence of toxicity when SEM-AVS<0 could not be rejected. However, the onset of Zn toxicity occurred at lower concentrations than generally assumed.

Single versus combined exposure of Hyalella azteca to zinc contaminated sediment and food.

The amphipod Hyalella azteca was exposed for 28 d to different combinations of Zn contaminated sediment and food. Sediment exposure (+clean food) resulted in increased Zn body burdens, increased mortality and decreased body mass when the molar concentrations of simultaneously extracted Zn were greater than the molar concentration of Acid Volatile Sulfide (SEM(Zn)-AVS>0), suggesting that dissolved Zn was a dominant route of exposure. No adverse effect was noted in the food exposure (+clean sediment), suggesting selective feeding or regulation. Combined exposure (sediment+food) significantly increased adverse effects in comparison with sediment exposure, indicating contribution of dietary Zn to toxicity and bioaccumulation. The observed enhanced toxicity also supports the assumption on the presence of an avoidance/selective feeding reaction of the amphipods in the single sediment or food exposures. During 14 d post-exposure in clean medium, the organisms from the same combined exposure history received two feeding regimes, i.e. clean food and Zn spiked food. Elevated Zn bioaccumulation and reduced reproduction were noted in amphipods that were offered Zn spiked food compared to the respective organisms that were fed clean food. This was explained by the failure of avoidance/selective feeding behavior in the absence of an alternative food source (sediment), forcing the amphipods to take up Zn while feeding. Increasing Zn body burdens rejected the assumption that Zn uptake from food was regulated by H. azteca. Our results show that the selective feeding behavior should be accounted for when assessing ecological effects of Zn or other contaminants, especially when contaminated food is a potential exposure route.

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.

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.

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.

Metallothioneins and cytosolic metals in Neomysis integer exposed to cadmium at different salinities.

In the present study the induction of metallothioneins (MTs) and its relation to cytosolic metal concentrations (Zn, Cu and Cd) in the euryhaline crustacean Neomysis integer exposed to Cd at different salinities was studied. N. integer was exposed to the same free cadmium ion activity of 5.74 x 10(-9) mol l(-1) (i.e. 1/5 of the 96 h LC(50) value expressed as cadmium activity) in hypo-osmotic (5 psu), isosmotic (16 psu) and hyper-osmotic media (25 psu) for 7 days. In this way, the effect of salinity on cadmium speciation was eliminated and therefore the physiological effect of salinity on Cd accumulation and MT induction could be studied. The accumulation of cytosolic Cd in N. integer changed with salinity from 1.11+/-0.05 micromol l(-1) at 5 psu up to 1.43+/-0.17 micromol l(-1) at 25 psu. This could indicate that the physiological response of euryhaline estuarine invertebrates like N. integer to salinity changes can influence the rate of trace metal uptake from solution. While the salinity changes did not cause significant differences in cytosolic Zn concentrations (mean value of all tested salinities: 34.4+/-2.8 micromol l(-1)), an inverse relationship between salinity and cytosolic Cu concentration was observed. The highest concentration of 15.7+/-2.3 micromol Cul(-1) was determined at 5 psu and the lowest 10.9+/-1.4 micromol Cul(-1) at 25 psu. This could point to a possible relationship between the copper concentration and the hemocyanin metabolism in N. integer. This is the first time that differential pulse voltammetry method was applied to MT assays with N. integer. Although the exposure to Cd resulted in a higher Cd cytosolic concentration, no subsequent MT increase was detected. The significant positive correlation between MT levels and cytosolic Cu concentrations (Spearman correlation coefficient r(s)=0.356, p=0.009) implies a strong relationship between MT and Cu in N. integer.

Comparison of nickel toxicity to cladocerans in soft versus hard surface waters.

The aims of the present study were to investigate (1) whether cladocerans living in soft water (operationally defined hardness < 10 mg CaCO(3)/L) are intrinsically more sensitive to Ni than cladocerans living in hard water (operationally defined hardness > 25 mg CaCO(3)/L) and (2) whether a single bioavailability model can be used to predict the protective effect of water hardness on the toxicity of Ni to cladocerans in both soft and hard water. To address these research questions, acute and chronic bioassays were conducted with 10 different cladoceran species collected in soft and hard water lakes in Sweden. Soft water organisms were tested in a 'soft' and a 'moderately hard' test water (nominal hardness = 6.25 and 16.3 mg CaCO(3)/L, respectively). Hard water organisms were tested in a 'moderately hard' and a 'hard' test water (nominal hardness = 16.3 and 43.4 mg CaCO(3)/L, respectively). The results of the toxicity tests in the 'moderately hard' test water revealed no significant differences between the intrinsic sensitivity of soft versus hard water organisms. Modeling exercises indicated that water hardness significantly reduced Ni toxicity to both the soft and the hard water organisms tested. Although predictions of chronic toxicity were sufficiently accurate using the same logK(CaBL) and logK(MgBL) (i.e. the model parameters describing the effect of hardness) for all organisms under consideration, predictions of acute toxicity were significantly more accurate when separate logK(CaBL) and logK(MgBL) values were derived for the soft and the hard water organisms tested. This is due to the fact that the relative decrease of acute Ni toxicity to soft water organisms in 'moderately hard' compared to 'soft' test water was significantly higher than for hard water organisms in 'hard' compared to 'moderately hard' test water.

Mechanisms of chronic waterborne Zn toxicity in Daphnia magna.

In order to gain better insights in the integrated response of Daphnia magna following chronic zinc exposure, several physiological parameters were measured in a time-dependent manner. D. magna juveniles were exposed for 21 days to dissolved Zn concentrations up to 340 microg/L. Next to standard endpoints such as mortality, growth and reproduction the following sub-lethal endpoints were measured: filtration and ingestion rate, respiration rate, energy reserves, internal Zn and total Ca concentrations in the organisms. Organisms exposed to 80 microg/L generally performed better than the Zn deprived control organisms. The former were used to elucidate the effects of higher Zn concentrations on the endpoints mentioned above. After 1 week, only 7% of the organisms exposed to 340 microg/L survived. Body Zn contents of these organisms were 281 +/- 76 microg g dry weight and a 37% decrease of the Ca contents was observed. This suggests a competitive effect of Zn on Ca uptake. Filtration rate (-51%), individual weight (-58%) and energy reserves (-35%) also exhibited a decreasing trend as a function of increasing Zn exposure concentrations. During the second and third exposure week an overall repair process was observed. In the surviving organisms mortality and reproduction were only slightly affected. This can be explained by (over)compensation reactions at lower levels of biological organisation: Ca contents (+24%) and filtration rate (+90%) increased as a function of the exposure concentration while respiration rate decreased (-29%) resulting in energy reserves remaining constant as a function of Zn exposure. It is hypothesized that a disturbed Ca balance is probably the first cause for zinc toxicity effects in D. magna.

Inter- and intra-species variation in acute zinc tolerance of field-collected cladoceran populations.

Acute zinc toxicity was assessed for 10 freshwater cladoceran species collected in six different ecosystems across Europe and for two standard laboratory-reared species (Daphnia magna and Ceriodaphnia dubia). The collected organisms belonged to five different genera: Daphnia (subgenus Daphnia and Ctenodaphnia), Ceriodaphnia, Simocephalus, Acroperus and Chydorus. The 48-h EC50 of the field-collected organisms tested in standard laboratory water ranged from 375+/-141 to 4314+/-1513 microg Znl(-1). The laboratory clone of D. magna was less sensitive than the majority of the field-collected species, while our laboratory Ceriodaphnia dubia was the second most sensitive. Considerable inter-species variation was found within the genus of Ceriodaphnia (factor 6) and within the genus Daphnia (factor 8). Among the different (sub)genera tested, Chydorus and Ctenodaphnia were significantly more tolerant than the others (up to a factor 3 difference). A significant positive relationship (r2=0.67, p<0.05) between the mean cladoceran 48-h EC50 and the ambient zinc concentration of the different aquatic systems was demonstrated, suggesting a role of acclimation and/or adaptation. No significant correlation between the acute zinc tolerance and the length of the organisms was found.

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.

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.

Multi-generation cadmium acclimation and tolerance in Daphnia magna Straus.

The cladoceran Daphnia magna was acclimated for seven generations to cadmium concentrations ranging from 0 (control) to 250 microg/l Cd (corresponding to a free ion activity of 4.60 nM Cd2+). Acute and chronic cadmium tolerance as well as cadmium accumulation were monitored as a function of acclimation time. After two to three generations of acclimation to concentrations ranging from 0.23 to 1.11 nM Cd2+ increases in acute tolerance were maximal (factor 7.2) and significant. Acclimation for seven generations to the same acclimation concentrations did result in an increased chronic cadmium tolerance (21 days EC50 values increased). Organisms acclimated to 1.93 nM Cd2+ were equally or more sensitive than non-acclimated daphnids in acute and chronic toxicity tests. Cadmium contents in D. magna increased significantly as a function of the acclimation concentration. Maximum body burdens of 236+/-30 microg Cd/g dry weight were measured in organisms exposed to 4.60 nM Cd2+, but detoxification mechanisms were only successful up to 82+/-20 microg Cd/g dry weight as this concentration did not cause major decreases in survival and reproduction in chronic toxicity tests. As the potential positive effect of acclimation on cadmium tolerance disappeared with successive acclimation generations and increasing acclimation concentrations, it is concluded that multi-generation acclimation studies are important for the evaluation of the long-term effects of environmental toxicants.

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.

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.