Environmental Toxicology and Chemistry, Vol. 35, No. 12, pp. 2987–2997, 2016 # 2016 SETAC Printed in the USA
A COMPARATIVE APPROACH USING ECOTOXICOLOGICAL METHODS FROM SINGLE-SPECIES BIOASSAYS TO MODEL ECOSYSTEMS ARNE HAEGERBAEUMER,*y SEBASTIAN HÖSS,z KAI RISTAU,y EVELYN CLAUS,x CHRISTEL MÖHLENKAMP,x PETER HEININGER,x and WALTER TRAUNSPURGERy yDepartment of Animal Ecology, University of Bielefeld, Bielefeld, Germany zEcological Sediment and Soil Assessment, Starnberg, Germany xGerman Federal Institute of Hydrology, Koblenz, Germany
(Submitted 6 January 2016; Returned for Revision 15 February 2016; Accepted 6 May 2016) Abstract: Soft sediments are often hotspots of chemical contamination, and a thorough ecotoxicological assessment of this habitat can help to identify the causes of stress and to improve the health of the respective ecosystems. As an important component of the ecologically relevant meiobenthic fauna, nematodes can be used for sediment assessments, with various assay tools ranging from single-species toxicity tests to field studies. In the present study, microcosms containing sediment were used to investigate direct and indirect effects of zinc on natural nematode assemblages, and acute community toxicity tests considering only direct toxicity were conducted. The responses of the various freshwater nematode species in both approaches were compared with those of Caenorhabditis elegans, determined in standardized tests (ISO 10872). At a median lethal concentration (LC50) of 20 mg Zn/L, C. elegans represented the median susceptibility of 15 examined nematode species examined in the acute community toxicity tests. In the microcosms, Zn affected the nematodes dose-dependently, with changes in species composition first detected at 13 mg Zn/kg to 19 mg Zn/kg sediment dry weight. The observed species sensitivities in the microcosms corresponded better to field observations than to the results of the acute community toxicity tests. Environ Toxicol Chem 2016;35:2987–2997. # 2016 SETAC Keywords: Sediment toxicity
Microcosms
Caenorhabditis elegans
Nematodes
Zinc
nutrient cycles [12–15] and in the remineralization of organic carbon [16]. Nematodes are thus excellent bioindicators for assessing the effects of contaminants in various habitats [17]. Specific nematode-based indices have been developed to reveal chemical disturbances in soils [18] and freshwater sediments [19]. The NemaSPEAR[%] index was developed as a stress index for freshwater sediments, derived from a large field dataset of nematode species sampled from rivers with a pollution gradient [20]. The co-occurrence approach (i.e., multivariate correlation of nematode species and environmental conditions in the same sample) aimed at identifying species that mainly occur in uncontaminated sediments (nematode species at risk ¼ NemaSPEAR) and species that are ubiquitous or also occur in polluted sediments (nematode species not at risk ¼ NemaSPEnotAR). Among the many nematode species, the soil nematode Caenorhabditis elegans [21] is by far the most commonly used for testing the toxicity of chemicals and environmental samples [22]. The interest in C. elegans as a model organism stems from the fact that it is easily cultured and has a simple body plan as well as a short life cycle. Moreover, the effects of numerous chemicals on this nematode at various organizational levels (molecular, organs, whole-organism, and population) have been described, and standardized test systems have been developed [23,24]. Even though C. elegans is a soil nematode, an additional advantage of this test organism is that testing can be carried out both in aqueous media and on solid substrates. The risks of chemicals in water, soil, and sediments can be determined with the same model organism [15,25]. The common use of single species as tools to assess the direct toxicity of chemicals is not only because of economic considerations, but also because of the valuable information
INTRODUCTION
A wide range of chemical compounds is discharged into freshwater ecosystems by industrial, agricultural, and other anthropogenic sources, and the contamination of sediments is a major environmental concern [1]. However, sediments are not only sinks for contaminants but also act as an important exposure route for aquatic organisms (e.g., after flood events) [2]. Thus, contaminants that have accumulated in sediments pose a possible threat to benthic species, but through processes such as remobilization and bioaccumulation also on the entire aquatic ecosystem. Accumulated metals within aquatic organisms can become a significant source for predators through trophic transfer [3]. Especially in soft (fine and sandy) sediments, meiobenthic taxa are more abundant and species-rich than macrobenthic invertebrates [4], although the latter are commonly used to assess the ecological quality of sediments [5]. Nearly all (95%) meiobenthic taxa are restricted to a free-burrowing lifestyle in aerobic sediment horizons and are therefore in contact throughout their life cycle with noxious substances in the sediment [6,7]. As a result, contaminated meiofaunal species represent a route to remobilize contaminants such as metals via sediment-based food webs [8]. Among meiobenthic communities, nematodes are the dominant organismal group in freshwater and marine sediments [9–11]. Furthermore, nematodes represent different trophic levels and occupy significant positions between micro- and macrofauna in the food web, in which they play an important role in This article includes online-only Supplemental Data. * Address correspondence to
[email protected] Published online 7 May 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.3482 2987
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these organisms provide, which allows the effects of the tested chemicals on individuals (e.g., toxicity thresholds and modes of action) to be predicted. However, the ecological relevance of single-species testing is limited, and the extrapolation of toxicity data from such tests to higher ecological levels is problematic [7,26]. In contrast to standardized toxicity tests with a single species (e.g., C. elegans), in community-based approaches (e.g., multispecies tests, model ecosystems) multiple species are tested under comparable exposure conditions (e.g., test concentration, test medium), allowing better comparisons of the responses of the different species to the chemicals of interest [27,28]. By first conducting acute community toxicity tests with free-living freshwater nematodes, indirect effects via the food web can be excluded so that the direct responses to certain chemicals can be compared, both among the observed species and between those species and standard test organisms. Moreover, by isolating natural nematode assemblages, even species that cannot be maintained in culture can be assessed. Acute community tests can therefore fill the gap between standard toxicity tests and microcosm studies. Complex, long-term, microcosm studies allow the inclusion of all matrix and indirect (food web) effects, thus mimicking real-life exposure scenarios [29]. The effects of chemicals on single nematode species and nematode communities in model ecosystems have been described [22], whereas much less is known about the direct toxicity of chemicals on free-living nematode assemblages [30–32]. The aim of the present study was to assess the response of natural nematode assemblages to chemical stress (Zn) after a long-term exposure in complex sediment microcosms, in which indirect food web effects could be evaluated, and in acute community toxicity tests, in which only the direct toxic effects of Zn were considered. The responses of the various freshwater nematode species in the 2 approaches were compared with the effects of Zn on C. elegans in single-species toxicity testing in aqueous medium and sediment. Using this comparative approach, a better understanding of nematode community responses to chemical stress and a more accurate interpretation of toxicity tests in terms of sediment quality assessment were expected. Specifically, in the present study, undisturbed nematode assemblages from pristine freshwater sediments were exposed to Zn, either in aqueous medium in acute community toxicity tests or in sediment in a microcosm setup. In addition, a standardized toxicity test with C. elegans was performed to define toxicity thresholds (x% effect concentration [ECx]) for Zn in sediments and aqueous medium, using survival and reproduction as toxicity parameters [15]. For the microcosms, Zn toxicity was assessed by measuring the Zn concentrations in the whole sediment, in the filtered porewater, and in the overlying water, and with accompanying C. elegans toxicity tests in whole sediment samples to estimate bioavailability and direct toxicity. MATERIALS AND METHODS
Sediment sampling and properties
Sediment used for the different toxicity tests (Supplemental € Data, Figure S1) was collected from the Ortze (538000 58.400 N, 0 00 10805 00.5 E), a sandy stream located in the Lunenburg Heath (Lower Saxony, Germany; 18% water content; 2.1% gravel, 96.1% sand, 1.5% silt and clay, 0.3% total organic carbon) characterized by low levels of anthropogenic contamination
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(metals, arsenic, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, hexachlorocyclohexane, DDTs, and hexachlorocyclobenzenes below the detection limits; Supplemental Data, Table S1). Stream water and the upper 5 cm of the surface sediment, including the indigenous benthic community, were collected with clean stainless scoops, transferred to high-density polyethylene containers, transported to the laboratory, and stored under refrigeration for 1 d until the start of the experiment following standardized guidelines [33,34]. C. elegans toxicity tests
Chronic toxicity tests with first-stage juveniles (J1) of the nematode C. elegans (N2 strain, Bristol, UK) were carried out according to ISO 10872 [23] and Traunspurger et al. [15]. Briefly, tests were conducted in polystyrene multiwells (12-well multidishes; VWR) containing 0.5 g of sediment (wet wt) or aqueous solution mixed with 0.5 mL of Escherichia coli (OP50) suspended in K-medium (3.1 g NaCl/L, 2.4 g KCl/L [35]) as food supply. Four replicates were set up for controls and each € concentration step. To assess sediment toxicity of Zn, dry Ortze sediment (dried at 105 8C for 5 h) was spiked with solutions of ZnCl2 (Riedel-de Haën, CAS 7846-85-7) in K-medium in 14 concentration steps, yielding nominal sediment concentrations ranging from 0 mg/kg to 320 mg/kg dry weight. To test the toxicity of Zn in aqueous medium, 8 stock solutions of ZnCl2 in K-medium were prepared, yielding final nominal test concentrations ranging from 0 mg/L to 64 mg/L. In both tests, 10 J1 juveniles were placed in each test vial and incubated at 20 8C in the dark for 96 h. The test was terminated by heat-killing the nematodes in a drying oven for 10 min at approximately 80 8C. After isolating the nematodes from the test medium according to standard procedures (ISO 10872 [23]), the number of offspring was counted and the inhibition of reproduction (%) was calculated in relation to the negative control (medium without Zn). For acute toxicity testing in aqueous medium, ZnCl2 solutions were prepared in K-medium (or K-medium without Zn for the controls) yielding 9 different nominal test concentrations of Zn ranging from 0 mg/L to 500 mg/L. Then 1 mL was transferred to each test well (12-well multi-dishes), setting up 4 replicates for control and each concentration step. Acute tests were conducted without food supply in accordance with standard guidelines [36,37]. The test wells were incubated at 20 8C in the dark for 48 h. Death was defined as a total lack of movement and a lack of response to probing with a needle. Acute community tests
The direct toxicity of Zn in aqueous medium was determined in acute community tests with in situ nematodes. Nematodes were extracted from pristine sediment following a modification of the centrifugal–flotation method of Higgins and Thiel [38], using a silica gel with a specific density of 1.14 g/cm3 (Ludox TM 50, Sigma-Aldrich). After centrifugation, the supernatant was filtered through a 35-mm sieve; the retained meiobenthic organisms, including various nematodes species, were thoroughly washed with K-medium to remove silica gel residues and kept in the same medium until use. Glass dishes (volume ¼ 20 mL, diameter ¼ 4 cm) were filled with 4 mL of K-medium (control) or a solution of ZnCl2 prepared in K-medium (nominal test concentration: ¼ 20 20 mg Zn/L; based on the median lethal concentration [LC50] for C. elegans). Four replicates were set up for control and Zn treatments. From the aqueous pool of extracted nematodes, 100 nematodes were randomly transferred into each replicate
Comparing bioassays from single-species tests to model ecosystems
dish. Vitality was checked by gentle probing with a needle. Only individuals responding to the mechanical stimulation were transferred to the test dishes. During the tests, the dishes were incubated at 20 8C in the dark. After 48 h, nematode vitality was checked by mechanical stimulation. Nematodes not responding to probing were considered dead. Dead and living individuals of each replicate were separately prepared in glycerine [39,40] and taxonomically identified to the species level following the method of Traunspurger [41], based on microscopy observations at 1250-fold magnification. Only species that showed 70% survival in the controls and occurred with 5 individuals in each treatment were considered for the sensitivity ranking. The survival in the treatments was adjusted by the survival in the controls according to Abbott [42], to exclude death from other causes than Zn. To compare acute effects of Zn on the nematode communities with effects on C. elegans, this species was tested at the nominal Zn concentration of 20 mg/L according to the method described in the previous section (C. elegans toxicity tests). Microcosm experiments
Setup and spiking. Collected sediment was homogenized with a stainless steel scoop and transferred in equal portions to 43 microcosms (glass jars; volume ¼ 1700 mL, diameter ¼ 12 cm) so that each contained approximately 800 g sediment (wet wt). For each of the 3 sampling dates, 4 replicates per treatment were set up. In addition, 7 untreated microcosms served to determine the initial sediment parameters. Stream water from the sampling site was carefully added immediately to minimize the exposure time to the air. Dry mass of the sediment was determined in accordance with ISO method EN 15934 [43]. After a 4-wk period of acclimatization (day 0), the overlying water was removed before adding Zn to the sediment of the microcosms. Sediment spiking was carried out, with slight modifications, according to standard procedures [44,45]. A 10-g (wet wt) sediment aliquot was taken from each microcosm and mixed thoroughly with 1 mL of the respective ZnCl2 stock solutions (Riedel-de Haën, CAS 7846-85-7, prepared in deionized water) or deionized water (control). After storing for 48 h at 20 8C to allow for partitioning of Zn from water to sediment particles, the spiked sediment aliquots were added to the microcosms and gently mixed for several minutes to ensure a homogeneous distribution of Zn within the sediment. This yielded nominal Zn sediment concentrations of 0 mg/kg (control), 10 mg/kg (low concentration), and 100 mg/kg (high concentration) dry weight. The highest Zn concentration was aligned with the 20% effect concentration (EC20) for C. elegans, revealed for reproduction in the sediment € toxicity tests with Ortze sediment. After that, the overlying water was carefully transferred back to the microcosms to obtain a recommended sediment–water volume ratio of approximately 1:2. The experiment was carried out under a 12:12-h light: dark regime (photon flow density of 15 mE/s m2) and constant environmental conditions (water aerated; water temperature, 20 2 8C; pH, 7.8 0.3). During the course of the experiment, evaporated water was replaced by a mixture (50:50) of deionized water and water collected from the sampling site. Sampling design. The nematode community was monitored over a period of 6 mo (180 d), with samples taken shortly before spiking (day 0) and after 30 d, 90 d, and 180 d (Supplemental Data, Figure S2). For analysis of biological and chemical
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parameters, the 4 replicate microcosms of each treatment and each sampling date were sacrificed. The overlying water column of each microcosm was gently removed with a suction pump and pooled for each treatment for analysis of Zn. A 500-mL sample of the overlying water was filtered through a glass-fiber filter (pore size: 1 mm; Pall), acidified with nitric acid (100 mL HNO3/100 mL) and stored at 8 2 8C until further analysis. For porewater extraction, 250-g (wet wt) subsamples of sediment were taken randomly from each replicate microcosm. Extraction of porewater followed standard procedures [45,46]: the sediment subsamples were centrifuged for 15 min at 4500 g, after which 50 mL of the supernatant was filtered through a cellulose nitrate filter (pore size: 0.45 mm; Sartorius), acidified with nitric acid (100 mL HNO3/100 mL), and stored at 8 2 8C until further analysis. For nematode community analysis, polyethylene corers with a diameter of 3.5 cm were used to randomly collect 3 sediment subsamples from each replicate microcosm, which were subsequently pooled. Before further processing, 50 g (wet wt) of the sediment subsamples of each treatment were separated for chemical analysis, and 10 g were used to test the direct toxicity of the whole sediment on C. elegans. To prevent contamination between the different treatments, all materials used were decontaminated according to standard protocols between samples [45]. Physicochemical parameters of the overlying water (pH, O2, electrical conductivity, and temperature), as well as the adenosine triphosphate (ATP) and chlorophyll a (Chl-a) contents of the sediments, as surrogate parameters for the biomass of bacteria and benthic algae, respectively, were measured every 30 d in microcosms maintained until the end of the experiment (180 d). Physicochemical parameters were measured with a calibrated multiparameter meter (Hanna HI 9828; Hanna Instruments). For measurements of Chl-a and ATP, 3 mL (2 1.5-mL subsamples each) of the uppermost sediment layer (0–1.5 cm depth) of each microcosm was sampled with a micropipette (Eppendorf Research 5000 mL) and immediately analyzed. The measurements followed general quality assurance requirements [47] and internal protocols. The ATP was measured according to the instructions included with the BacTiter-GloTM microbial cell viability assay (Promega), and the results were then converted into bacterial biomass by applying a conversion factor of 297 ATP (mg ATP) ¼ 1 mg C [48]. Samples used to determine the biomass of benthic algae were centrifuged for 5 min at 2000 g. The supernatant was discarded, and 10 mL of 90% ethanol was added to the sediment pellet. The sample was then incubated overnight at 4 8C in the dark to extract Chl-a [49], followed by a centrifugation step for 10 min at 3000 g. The Chl-a concentration in the supernatant was measured spectrophotometrically, with a correction for pheopigments by acidification [50]. After both ATP and Chl-a had been measured, each sediment pellet was weighed, and the bacterial and algal biomasses were normalized to sediment dry weight using the previously € measured dry weight:wet weight ratio of 1:1.18 for the Ortze sediment used. Chemical and toxicity analysis of whole sediment, porewater, and overlying water. Total whole-sediment concentrations of Zn were measured externally at the German Federal Institute of Hydrology (Koblenz, Germany) via aqua regia dissolution and inductively coupled plasma–optical emission spectrometry (ICP–OES) in accordance with standardized methods [51]. For each sampling date, the filtrates obtained of the extracted
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porewater and the overlying water were acidified and analyzed by inductively coupled plasma–mass spectrometry (ICP–MS) at the German Federal Institute of Hydrology. In addition, the direct toxicity of whole-sediment samples from the microcosms on C. elegans was tested according to standard procedures (ISO 19872; see also the C. elegans toxicity tests section). Nematode community analysis. The sediment samples for the nematode community analysis were fixed with formalin (4% v/v) and stained with Rose Bengal. Nematodes were extracted from the sediment as described in the previous section (Acute community tests). All individuals in each sample were counted under a stereomicroscope (40-fold magnification; Olympus SZ40). The first 50 nematodes were mounted on slides according to the method of Seinhorst [39,40], identified to the species level (1250-fold magnification), and classified into feeding type: deposit feeders (mainly feeding on bacteria), epistrate feeders (mainly feeding on algae), suction feeders, and chewers (predatory/omnivorous nematodes), based on the morphological attributes of their buccal cavity [41]. In the nematode community analyses, abundance, and species richness (S), as univariate parameters, were calculated for the different treatments and sampling dates. Nematode abundances were log (xþ1)–transformed to meet homogeneity of variance assumptions. Changes in nematode species composition were evaluated with principal response curves, calculated with CANOCO (Ver 4.55; Biometris-Plant Research International,). This multivariate technique uses an ordination method based on a redundancy analysis [29] to compare the species composition response in Zn-spiked sediments with that of control communities over time. The interactions between sampling dates and the Zn treatment served as an explanatory variable, and time served as a covariable. A linear combination of variables was calculated to determine the deviation of each species composition from treated to control treatments and expressed as the first principal component of the variance explained by treatment differences in time (cdt). The principal response curves were derived by plotting the cdt against the time, representing the course of the 2 treatments (low concentration, high concentration) versus time relative to the control treatment. The corresponding species scores (bk) allow interpretations of the principal response curves at the species level. The higher the bk value of a species, the more the actual response pattern of the species was likely to follow the principal response curve pattern. Species with negative values were inferred to show the opposite pattern, whereas species with scores close to 0 presumably had no response or a response unrelated to the principal response curve pattern. The NemaSPEAR[%]metal of the treatments was determined by calculating the relative abundance (%) of nematode species classified as SPEAR (species at risk) for metal pollution with respect to the relative abundance of all species [19]. The relative abundances of very dominant species were log (xþ1)–transformed. NemaSPEAR½%metal ¼ 100
S log ½NemaSPEARrelAb S log ½AllSpeciesrelAb
The terms log [NemaSPEAR]relAb and log [AllSpecies]relAb are the logarithms of the relative abundances of nematode species at risk and of all identified species in the treatments, respectively.
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Statistical data analysis
Data from the C. elegans toxicity tests and from experiments on the effects of the spiked sediment and the aqueous ZnCl2 solutions were statistically analyzed using a one-way analysis of variance (ANOVA) followed by a 2-sided Dunnett post hoc comparison using SPSS (Ver 17.0). The ECx values for Zn in both media were calculated by fitting the toxicity data (% inhibition reproduction; % mortality) using a sigmoidal, logistic model and SigmaPlot (Ver 11.0; Systat) to achieve concentration–response curves, from which the EC20 and median effect and lethal concentrations (EC50 and LC50, respectively) were calculated. The physicochemical and biological (ATP and Chl-a measurements) parameters of the microcosms were analyzed statistically by a repeated measures analysis of variance (RMANOVA) and a post hoc comparison (Tukey’s honest significant difference [HSD] test). If the assumption of sphericity was not met, a Greenhouse–Geisser correction and the Bonferroni post hoc test were conducted. In case of general significant differences between treatments or sampling dates, a one-way ANOVA followed by a post hoc comparison using Tukey’s HSD test was performed separately for every sampling date. Differences in the Zn concentrations of the particular phases (sediment, porewater) of the microcosms between the various treatments were analyzed statistically with a one-way ANOVA followed by a post hoc Tukey’s HSD test. Changes in the univariate parameters and feeding-type composition between treatments and over time were evaluated with a two-way ANOVA, followed by a post hoc comparison (Tukey’s HSD). If general significant differences between treatments or sampling dates were determined, a one-way ANOVA followed by a post hoc comparison (Tukey’s HSD) was performed. The statistical significance of the explanatory variables regarding nematode species composition was tested with a Monte Carlo permutation test based on the redundancy analysis from which the principal response curves were derived [29]. RESULTS
Zn toxicity to C. elegans
The effects of Zn in spiked sediments and aqueous medium on reproduction were clearly dose dependent (Figure 1), with significant effects compared with control first being observed at a nominal concentrations of 80 mg/kg dry weight and 4 mg/L for sediment and aqueous medium, respectively (p < 0.001, one-way ANOVA, post hoc Dunnett). In the acute toxicity tests, significant effects on survival compared with control first occurred at a nominal concentration of 10 mg Zn/L (p < 0.001, one-way ANOVA, post hoc Dunnett). The toxicity thresholds (ECx values) determined are provided in Table 1. Acute community tests
The toxicity test was valid in terms of more than 90% survival of the tested individuals in the control according to quality assurance requirements and standardized guidelines [36,37]. In terms of the various species, 15 of the 86 identified species met the criteria for inclusion in the sensitivity comparison (5 individuals per treatment; 70% species survival in the control; Supplemental Data, Table S2). The survival of C. elegans exposed to 20 mg Zn/L (nominal concentration) was added to the sensitivity ranking as reference value.
Comparing bioassays from single-species tests to model ecosystems
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Figure 1. Concentration–response curves of Zn toxicity for Caenorhabditis elegans exposed to spiked sediment (A) and aqueous medium (B; filled circles ¼ reproduction; triangles ¼ mortality), determined as percent inhibition of reproduction compared with control (A, B) and percent mortality (B) as toxicity endpoints (mean standard deviation, n ¼ 4). Table 1. Low and median effect concentrations of Zn, calculated for reproduction and survival of Caenorhabditis elegans Test matrix Sediment Aqueous medium
Unit
Toxicity endpointa
EC20b
EC/LC50b
mg/kg dry wt mg/L
Reproduction Reproduction Survival
94.7 28.9 2.3 0.1 n.d.
162.1 48.8 3.4 0.1 19.9 5.3
Exposure time: reproduction ¼ 96 h; survival ¼ 48 h. Nominal concentrations (mean standard deviation). EC20 ¼ 20% effect concentration; EC/LC50 ¼ median effect and lethal concentration; n.d. ¼ not determined. a
b
The nematode species analyzed clearly differed in their sensitivity to the exposed nominal Zn concentration of 20 mg/L (Table 2). Whereas Epitobrilus stefanskii and Dorylaimus stagnalis were tolerant over the test duration of 48 h, with an adjusted survival of >85% in the Zn treatment, species such as Tripyla setifera, Eumonhystera filiformis, Ironus macramphis, and Anaplectus granulosus were sensitive to Zn, as evidenced by a survival rate that was 0.05, RMANOVA). The water temperature remained relatively constant over time (19.7–22.6 8C; Table 3), independent of the treatment (p > 0.05, RMANOVA). The conductivity varied between 160 mS/cm and 390 mS/cm, with significant differences between the control and high-concentration treatments (p < 0.05, RMANOVA, post hoc Tukey’s HSD; Table 3). Within the treatments, a significant increase in the ATP content over time was recorded (p < 0.05, RMANOVA, post hoc Tukey’s HSD; Table 3). Overall, the biomass of benthic bacteria was significantly lower in the high-concentration microcosms than in either the control treatments (p < 0.01, RMANOVA, post hoc Tukey’s HSD; Table 3) or the lowconcentration microcosms (p < 0.05, RMANOVA, post hoc Tukey’s HSD; Table 3). The Chl-a concentrations in sediments changed significantly over time (p < 0.05, RMANOVA, post hoc Tukey’s HSD; Table 3), but there were no significant effects between the different treatments (p > 0.05, RMANOVA). Zn analysis and toxicity testing. At all sampling dates, the measured Zn concentrations in the microcosm sediments matched the target concentrations. A clear treatment-related regime with significant differences between the 3 treatments was determined (all p < 0.001, one-way ANOVA, post hoc Tukey’s HSD; Table 4). The Zn concentrations in the porewater and overlying water were 2 to 3 orders of magnitude below the sediment concentrations, but there was still a clear gradient between treatments and significant differences between the high-concentration treatment and the 2 other treatments (p < 0.01, one-way ANOVA, post hoc Tukey’s HSD; Table 4). The standardized toxicity test with C. elegans showed no toxic effects for the sediments of the low-concentration treatments at any sampling date (10% inhibition of reproduction; Table 4). In sediments from the high-concentration
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Table 3. Summary of the physicochemical parameters and ATP and Chl-a measurements in the microcosmsa C Dayb 30
LC
Day 90
Day 180
Day 30
HC
Day 90
Day 180
Day 30
Day 90
Day 180
pH 7.9 0.1 8.2 0.2 7.9 0.1 7.9 0.1 8.0 0.1 8.0 0.2 7.5 0.2 7.8 0.3 7.9 0.1 O2 (mg/L) 7.2 0.6 6.2 0.7 6.4 0.6 6.8 0.5 6.4 0.3 6.7 0.5 6.7 0.4 6.6 0.5 6.7 0.1 Cond. (mS/cm) 194.0 5.3 187.8 8.7 183.4 22.0 211.8 45.0 216.8 11.5 237.5 27.4* 279.0 15.4* 237.5 27.4* 218.0 9.2* Water temp. (8C) 20.1 0.4 22.0 20.5 21.4 0.3 20.1 0.1 21.9 0.9 21.3 0.2 20.8 0.2 21.9 0.7 21.7 0.1 ATP (ng C/g dry wt) 0.35 0.36 10.3 20.5 3997 3217 0.04 0.02 2.9 5.7* 1640 1634* 0.04 0.04 0.0 0.0* 184 238* Chl-a (mg/g dry wt) 1.14 0.45 0.97 0.29 0.67 0.16 1.19 0.11 0.85 0.35 0.60 0.13 1.46 0.29 0.95 0.3 0.86 0.22
Data are mean standard deviation, n ¼ 4. “Day” represents time after application. *Significantly different from control, p < 0.05, one-way ANOVA, post hoc Dunnett’s test. ATP ¼ adenosine triphosphate; Chl-a ¼ chlorophyll a; C ¼ control; LC ¼ low Zn concentration; HC ¼ high Zn concentration; Cond. ¼ electric conductivity.
a
b
microcosms, reproduction was significantly inhibited compared with either the control or the low-concentration microcosms at all sampling dates, with decreasing toxicity over the course of the experiment (p < 0.01, one-way ANOVA, post hoc Tukey HSD). Nematode community analysis. Overall, 1740 individuals belonging to 46 species were identified in the microcosm experiment. Prior to the addition of Zn (day 0), slight variations in the nematode communities of the untreated sediment were determined with respect to nematode abundances (34–65 individuals/100 mL sediment), number of species (6–11), NemaSPEAR[%]metal (21–44%), and feeding type composition (24–96% predators/omnivores; Table 5). Control communities changed over the course of the experiment, with increasing abundances (peaking at 90 d), slightly increasing NemaSPEAR[%]metal values, and a distinct shift from a predator/omnivore community to one dominated by deposit feeders (Table 5). The number of identified species was relatively constant. The presence of Zn reduced nematode abundance significantly in the high-concentration microcosms compared with either the control or the low-concentration microcosms over the entire experiment (p < 0.001, two-way ANOVA, post hoc Tukey’s HSD), whereas no differences were observed between the control microcosms and those with the lowest Zn dose. In terms of species richness, differences between the treatments were measured, with significantly fewer species in the high-concentration treatments than in the control or low-concentration treatments beginning on day 90 after Zn application (p < 0.001, two-way ANOVA, post hoc Tukey’s HSD).
The initial NemaSPEAR[%]metal value was 35.2%. After Zn addition, the values decreased dose-dependently and were significantly lower in the high-concentration microcosms than in the control and low-concentration microcosms (p < 0.05; two-way ANOVA; post hoc Tukey’s HSD). Regarding the feeding-type composition of the nematode community, initially approximately 76% of the individuals analyzed were predatory or omnivorous nematodes (Table 5 and Supplemental Data, Table S3). In the control and lowconcentration microcosms, the relative abundance of predacious and omnivorous species decreased significantly over the course of the experiment, to 16% and 38%, respectively (p < 0.05, one-way ANOVA, post hoc Tukey’s HSD). By contrast, deposit and epistrate feeders increased concurrently, up to 32% and 42% respectively, whereas the proportion of predators and omnivores in the high-concentration microcosms increased over time, to >90% from day 30 until the end of the experiment. Overall, the ratio of deposit and epistrate feeders to predatory and omnivorous nematodes increased significantly in the highconcentration microcosms compared with the control and lowconcentration microcosms (p < 0.001, two-way ANOVA, post hoc Tukey’s HSD). The nematode species composition was dose-dependently affected by the presence of Zn, with significant differences between treated and control microcosms. The principal response curves indicated significant differences between the different treatments with the first explanatory variable explaining 59% of the variance (Figure 2). Slight, but significant Zn-induced community alterations occurred in the low-concentration microcosms 180 d after the addition of Zn (p < 0.01, Monte
Table 4. Nominal and measured Zn concentrations in various phases of the microcosms and toxicity of whole sediment samples to Caenorhabditis elegansa C
Sediment (mg/kg dry wt) Nominal Measured Porewater (mg/L) Overlying water (mg/L) % I Repro C. elegansc a
LC
HC
Dayb 30
Day 90
Day 180
Day 30
Day 90
Day 180
Day 30
Day 90
Day 180
0 3.1 0.022 0.005 0
0 5.0 0.016 0.025 0
0 3.1 0.007 0.003 0
10 19.3* 0.032 0.019 0
10 13.0* 0.025 0.016 10
10 13.2* 0.024* 0.005 0
100 127.0* 2.110* 0.190* 67*
100 85.0* 1.010* 1.100* 20*
100 86.6* 0.223* 0.004 10
Replicate microcosms were pooled. “Day” represents sampling day. Percent inhibition of reproduction (% I Repro) of C. elegans after 96 h of exposure to LC and HC sediments compared with control sediments (ISO 10872 [23]); negative values (stimulation) were not regarded as toxic effects and were set to “0.” *Significantly different from control, p < 0.05, one-way ANOVA, post hoc Dunnett’s test. C ¼ control; LC ¼ low Zn concentration; HC ¼ high Zn concentration. b c
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Table 5. Summary of measured nematode community parameters in the microcosms Treatment C 180 LC HC
Sampling day
2993
a
Abundance (ind./100 mL sediment)
No. of species
NemaSPEAR[%]metal
Predators and omnivores (%)
48.4 10.5 46.3 18.6 95.9 43.9 7.3 1.3 31.1 14.9 149.6 196.8* 59.3 68.4 10.5 3.3* 13.6 2.4* 14.4 7.5*
7.6 1.7 6.5 0.6 7.4 0.5 46.7 7.0 6.0 1.6 8.5 2.9 5.3 1.3 4.5 1.3* 3.5 1.3* 2.5 1.3*
35.2 8.8 33.8 4.7 41.5 10.9 15.6 6.5 23.8 13.2 51.2 14.0 57.2 19.2 13.2 9.4* 27.0 18.9* 27.9 25.3*
76.2 24.9 81.5 9.9 52.6 18.4
0 30 90 65.6 9.7 30 90 180 30 90 180
92.2 11.0 48.4 35.1 37.7 41.0 90.0 2.7 95.4 4.7* 92.1 7.5*
a Data are mean standard deviation, n ¼ 4. *Significantly different from control, p < 0.05, one-way ANOVA, post hoc Dunnett’s test. NemaSPEAR ¼ nematode species at risk; C ¼ control; LC ¼ low Zn concentration; HC ¼ high Zn concentration.
Figure 2. Principle response curves (component of the variance explained by treatment difference in time [cdt]) resulting from the comparison of nematode communities in Zn-treated microcosms (first explanatory variable 59%, eigenvalue 0.197). The accompanying species scores (bk) indicate the species with the greatest contribution to the principal response curve pattern (0.2 < bk < 0.2 not shown; ** ¼ significantly different from control, p < 0.01, Monte Carlo permutation test).
Carlo permutation test; Figure 2). The clearest effects, however, were in the high-concentration treatments, where distinct deviations from the control were observed over the entire duration of the experiment (p < 0.01, Monte Carlo permutation test; Figure 2). The species scores indicated the species that considerably contributed to the deviations of the principal response curves from the control curve. Negative scores, indicating a decrease in abundance, were obtained for species of the bacterivorous genus Eumonhystera (E. vulgaris, E. pseudobulbosa, and E. longicaudatula), with E. vulgaris being most strongly affected by the Zn treatments. Tripyla setifera, an omnivorous nematode, had the most positive species score, indicating that it benefited from the Zn treatment. DISCUSSION
Acute toxicity studies are usually conducted with single species cultured under ideal conditions (diet, temperature, light, etc.) and tested in defined media with constant exposure to the compound of interest. In the present study, nematodes isolated from an intact in situ community from pristine sediment were used as test individuals, which allowed a more realistic scenario than can be obtained with cultured model organisms. The soil nematode C. elegans, an established model organism in ecotoxicological tests, served as the benchmark in this multispecies approach. Exposed to 20 mg Zn/L (nominal
concentration) in the acute toxicity test, C. elegans had a survival rate of 65%, showing an intermediate sensitivity to Zn among the 15 tested freshwater nematode species (9 species were more sensitive, and 6 species were less sensitive). Although nematodes belonging to the family Rhabditidae, such as C. elegans, are generally thought to be relatively tolerant of toxicants [18,52], Boyd and Williams [53] found that, compared with 2 other nematode species, C. elegans had an intermediate sensitivity, which is in agreement with the results of the present study. Given that species more chemically sensitive than C. elegans will not be protected based on estimates of the risk posed by chemicals, as determined in single-species toxicity tests, the extrapolation of toxicity data from single to multiple species remains problematic. The sensitivities of the tested nematode species were expected to be highly species-specific, in light of the very high functional and structural diversity of this taxon. For those species classified in the NemaSPEAR[%] index [19], the sensitivities determined in the acute community test were only partly in line with the classifications. For example, the relatively high sensitivity of Anaplectus granulosus, T. setifera, and Monhystrella paramacrura conflicts with their classification as nematode species not at risk (NemaSPEnotAR), resulting from their ubiquitous occurrence under diverse environmental conditions [14]. However, only 2 of the 15 ranked species are classified as NemaSPEAR. Thus, comparisons with field data
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should be made with caution, because the ranking obtained might represent only the so-called tolerant part of the complete range of nematode sensitivities as a result of the artificial test conditions. Nevertheless, both Aporcelaimellus obtusicaudatus and Ironus ignavus (both NemaSPEAR) were significantly more sensitive than other NemaSPEnotAR, such as D. stagnalis, E. stefanskii, and Panagrolaimus rigidus (Table 2), which is in line with the NemaSPEAR[%] index categories. Even though Zn is one of the most common anthropogenically introduced trace metals in the environment and the suitability of nematodes as test organisms is generally acknowledged, very little is known about the sensitivity of free-living freshwater nematodes to this contaminant [22]. Species of the Dorylaimina have been categorized as most sensitive [54,55], but we were able to confirm these findings only in A. obtusicaudatus, not in D. stagnalis. Kammenga et al. [30] examined the sensitivity of 12 different nematode species extracted from soil and exposed to Cd in acute tests. Both C. elegans and D. stagnalis showed an intermediate sensitivity and Cephalobus persegnis a high sensitivity toward this metal. In the present study, D. stagnalis was Zn tolerant, with >98% of the individuals still vital after 48 h of exposure compared with control, whereas both C. elegans and C. persegnis had a low to medium sensitivity toward Zn. The results of the acute community toxicity test were used to rank various freshwater nematode species according to their susceptibility to Zn and to directly compare the sensitivity of a mixed nematode assemblage with a standard test organism, excluding confounding factors imposed by sediment particles and other benthic food web components. However, the exclusion of these factors accompanied by the rather artificial test circumstances impeded comparisons with data from field conditions. The test setup in aqueous medium without food supply might overestimate the Zn tolerance of the species analyzed and lead to a shift toward species that are able to survive the artificial experimental conditions, even in the controls. This might explain the poor accordance of the ranking of acute susceptibilities with the field-derived NemaSPEAR classifications. Therefore, in addition, the response of nematode communities to Zn was studied in microcosms, where, more or less, all natural factors were included, more species could exist under these laboratory conditions, chronic exposure was also addressed, and experimental conditions could still be controlled. The microcosm study revealed clear dose-dependent effects of Zn on the nematode communities of freshwater sediments, with species composition as the indicator responding most sensitively to the impact of Zn. Multivariate analyses detected significant changes in the low-concentration microcosms, whereas inhibitory effects of Zn on abundance, species richness, feeding type composition, and the NemaSPEAR[%] index occurred only in the high-concentration treatments (Figure 2 and Table 5). In terms of species composition, very clear impacts of Zn on the high-concentration microcosms were already discerned by the first sampling date and increased until the end of the study. In the low-concentration microcosms, in contrast, Zn-induced alterations of species composition were observed only on the last sampling date (after 180 d). The effects of Zn on the nematode community could be predicted by considering the sediment toxicity on C. elegans, tested for all microcosm samples. Significant effects on the reproduction of C. elegans first occurred only at the highest Zn concentration (Table 4). Significant effects on the reproduction of C. elegans at sediment concentrations > 100 mg/kg dry weight were expected, however, given the EC20reproduction of 95 mg/kg dry
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weight (Table 1). The decrease in toxic effects of the highconcentration sediments over time (Table 4) can be best explained by the decreasing Zn concentrations in the porewater of the microcosms, assuming that porewater concentrations better predict the bioavailability of Zn than whole-sediment concentrations (Table 4). Regarding the toxicity thresholds of 2.3 mg Zn/L (EC20reproduction) and 3.4 mg Zn/L (EC50reproduction), as determined in chronic toxicity tests with C. elegans in aqueous medium, toxicity-relevant concentrations were measured after 30 d (2.1 mg/L), but the concentrations declined considerably to below the toxicity thresholds after 180 d (0.2 mg/L). Although the ECx values have to be regarded with caution, as they were based on nominal Zn concentrations, this latter finding indicates that the bioavailable Zn fraction decreased over the course of the experiment. Zinc sediment concentrations in the microcosms were comparable to those in river sediments with low to moderate contamination [19], even in the high-concentration microcosms (Table 4). However, the strong effects on the nematode communities in those microcosms might have been the result of comparably high porewater concentrations, especially within the first 30 d of the experiment (Table 4). Porewater concentrations were considerably higher than the concentrations expected in the field. Assuming an empirically derived solids–water partition coefficient of 122 L/g [56], maximal porewater concentrations of 0.001 mg Zn/L were predicted. The steadily decreasing porewater concentrations (Table 4) indicated that Zn partitioning did not reach equilibrium until 180 d. Moreover, experimental conditions (oxidized sediment) and specific sediment characteristics (percentage of sand, low total organic carbon) might have caused a relatively high bioavailability of Zn, leading to comparably strong effects at low whole-sediment concentrations. Because of the lack of freshwater microcosm studies, the results found in the present study were compared with effects of Zn on nematode communities in estuarine and soil microcosms. Distinct effects on the species composition of nematode communities inhabiting an estuary sand occurred at the lowest tested concentration (593 mg/kg dry wt) [57]. Korthals et al. [58,59] reported significant effects of Zn on soil nematode communities, with the strongest effects, observed within 2 wk, on the trophic structure (100 mg/kg soil dry wt), followed by total nematode abundance (200 mg/kg soil dry wt) and the maturity index (400 mg/kg soil dry wt). Smit et al. [60] reported Zn-induced effects on nematode species composition at 100 mg/kg to 320 mg/kg dry soil, but the communities recovered by the end of the study. A long-lasting alteration of the species composition was induced at 560 mg/kg dry soil. Thus, compared with estuarine and soil systems, freshwater nematode communities seem to be relatively sensitive to Zn, with first effects on the species level occurring at 20 mg/kg dry weight. However, comparisons with other studies based on bulk concentrations have to be made with caution, as the bioavailability of Zn, strongly influencing toxic effects, may have varied considerably in the various studies. In particular, the positive species score of T. setifera indicated the increased abundance of this omnivorous species in the treated microcosms, whereas bacterial feeding species of the genus Eumonhystera (E. vulgaris, E. pseudobulbosa, and E. longicaudatula) had negative scores (Figure 2). These findings are in accordance with the results of other microcosm experiments in which the effects of cadmium and ivermectin on nematode communities were investigated [32,61,62]. Thus, the chemically induced shift from bacterivor-dominated to predator
Comparing bioassays from single-species tests to model ecosystems
and omnivore–dominated nematode communities (Table 5) seems to be a general response to chemical stress, rather than a specific response to Zn. This is also confirmed by field observations, in which river sediments with mixed chemical contamination were dominated by omnivorous and predacious nematodes, mainly Tobrilidae (e.g., Tobrilus) and Mononchus species [19,20]. The hypothesis that the benefit of predacious nematodes in contaminated sediments was the result of the chemically induced extinction of macrobenthic competitors [20] could not be verified by these microcosm experiments, as macrobenthic organisms, and the indirect food web effects related to them, were excluded from the experiments. The good agreement between the microcosm results of the present study and field observations confirms the general suitability of the NemaSPEAR[%] index, derived from river data on chemical contamination and the co-occurring nematode fauna, as a metric of chemical disturbance in benthic habitats [19]. Indeed, the NemaSPEAR[%]metal was significantly lower in the high-concentration microcosms than in the control over the entire course of the experiment. As reported for cadmium and ivermectin [19], Zn similarly imposed dose-dependent effects on the NemaSPEAR[%] index. One of the aims of the present study was to reveal differences between the direct toxicity of Zn on nematode individuals and Zn-induced effects that occur via indirect pathways (e.g., through the food web). The toxicity of the Zn-spiked sediments from the microcosms on C. elegans (Table 4) indicated that Zn adversely affected the nematodes and thus may have been at least partly responsible for the observed community response. However, the shifts in the nematode community structure in the microcosms that could not be explained by the species sensitivity ranking determined by the acute community toxicity testing confirmed that other factors added to the direct toxicity of Zn (i.e., indirect toxicity). Concentrations of ATP, as a measure of bacterial biomass, increased significantly throughout the microcosm experiment and in each treatment, with the short-term decreases at the beginning of the experiment probably occurring as a result of the mechanical manipulation required during the microcosm setup [63] (Table 3). Compared with the low-concentration and control treatments, the ATP concentrations in highconcentration microcosms increased to a significantly lesser extent, resulting in dramatically lower bacterial biomasses compared with control values (only 5% at the end of the study; Table 3). This might explain why in high-concentration microcosms deposit feeders were not able to increase in abundance over the course of the experiment, as they did in the control (Supplemental Data, Table S3). These results suggested indirect bottom-up effects affecting the nematode community via Zn toxicity on bacteria. In contrast to ATP concentrations, Chl-a concentrations, as a measure of benthic algal biomass in the sediments, decreased significantly over time, and were not affected by the Zn treatment (Table 3). The decrease in algal biomass, however, did not influence the abundance of epistrate feeders among the nematodes (Supplemental Data, Table S3). CONCLUSIONS
The present study demonstrated a relatively large range in the Zn susceptibility of freshwater nematode species. The Zn sensitivity of the model organism C. elegans, used for standard toxicity testing (ISO 10872 [23]), appeared to be well within the range of the freshwater nematodes studied. However, the sensitivity ranking only partly agreed with field observations,
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probably because acute community testing in aqueous medium might only reveal species ranking among the more tolerant species (i.e., those able to survive the artificial experimental conditions), even in the controls, and because factors influencing the bioavailability of toxicants and indirect effects via the food web, such as occur in the field, were excluded from the acute community tests. We verified these hypotheses in a microcosm experiment with more realistic exposure conditions, including a sediment matrix and an intact meiobenthic organism assemblage. Zinc already affected species composition at sediment concentrations of 13 mg/kg to 19 mg/kg dry weight and porewater concentrations of 0.02 mg/L to 0.03 mg/L, indicating the presence of considerably more sensitive species in the microcosms than in the acute community tests. Moreover, besides the direct toxicity of Zn on the nematodes, the results of the microcosm experiment suggested indirect bottom-up effects via Zn toxicity on bacteria. Testing the toxicity of Zn under more realistic exposure conditions revealed a species sensitivity ranking comparable to field observations, which is also reflected by the dose-dependent response of the NemaSPEAR[%] index. Our results stress the importance of considering indirect effects in toxicity testing and confirm the value of nematodes as bioindicators, providing tools covering the whole range of environmental relevance, from single species to complex field studies. Supplemental Data—The Supplemental Data are available on the Wiley Online Library at DOI: 10.1002/etc.3482. Acknowledgment—We are grateful to the German Federal Institute of Hydrology (BfG), which funded the preparation of the present study as part of the project “Validation of the NemaSPEAR[%]-index based on field studies and laboratory experiments” (project M39620004003), and the technical support of S. Gehner (University of Bielefeld, Bielefeld, Germany) and M. Sturm and D. Fahrenkrog (BfG, Koblenz, Germany). We also thank P.M. Chapman and the 3 anonymous reviewers for their comments and editorial work on the manuscript. Data availability—Data are available from the corresponding author at
[email protected]
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