Replicate DNA metabarcoding can discriminate seasonal and spatial abundance shifts in river macroinvertebrate assemblages.

The delivery of consistent and accurate fine-resolution data on biodiversity using metabarcoding promises to improve environmental assessment and research. Whilst this approach is a substantial improvement upon traditional techniques, critics note that metabarcoding data are suitable for establishing taxon occurrence, but not abundance. We propose a novel hierarchical approach to recovering abundance information from metabarcoding, and demonstrate this technique using benthic macroinvertebrates. To sample a range of abundance structures without introducing additional changes in composition, we combined seasonal surveys with fish-exclusion experiments at Catamaran Brook in northern New Brunswick, Canada. Five monthly surveys collected 31 benthic samples for DNA metabarcoding divided between caged and control treatments. A further six samples per survey were processed using traditional morphological identification for comparison. By estimating the probability of detecting a single individual, multispecies abundance models infer changes in abundance based on changes in detection frequency. Using replicate detections of 184 genera (and 318 species) from metabarcoding samples, our analysis identified changes in abundance arising from both seasonal dynamics and the exclusion of fish predators. Counts obtained from morphological samples were highly variable, a feature that limited the opportunity for more robust comparison, and emphasizing the difficulty standard methods also face to detect changes in abundance. Our approach is the first to demonstrate how quantitative estimates of abundance can be made using metabarcoding, both among species within sites as well as within species among sites. Many samples are required to capture true abundance patterns, particularly in streams where counts are highly variable, but few studies can afford to process entire samples. Our approach allows study of responses across whole communities, and at fine taxonomic resolution. We discuss how ecological studies can use additional sampling to capture changes in abundance at fine resolution, and how this can complement broad-scale biomonitoring using DNA metabarcoding.

Towards generalised reference condition models for environmental assessment: a case study on rivers in Atlantic Canada.

Evaluation of the ecological status of river sites in Canada is supported by building models using the reference condition approach. However, geography, data scarcity and inter-operability constraints have frustrated attempts to monitor national-scale status and trends. This issue is particularly true in Atlantic Canada, where no ecological assessment system is currently available. Here, we present a reference condition model based on the River Invertebrate Prediction and Classification System approach with regional-scale applicability. To achieve this, we used biological monitoring data collected from wadeable streams across Atlantic Canada together with freely available, nationally consistent geographic information system (GIS) environmental data layers. For the first time, we demonstrated that it is possible to use data generated from different studies, even when collected using different sampling methods, to generate a robust predictive model. This model was successfully generated and tested using GIS-based rather than local habitat variables and showed improved performance when compared to a null model. In addition, ecological quality ratio data derived from the model responded to observed stressors in a test dataset. Implications for future large-scale implementation of river biomonitoring using a standardised approach with global application are presented.

Structural and functional responses of benthic invertebrates to imidacloprid in outdoor stream mesocosms.

Structural and functional responses of a benthic macroinvertebrate assemblage to pulses of the insecticide imidacloprid were assessed in outdoor stream mesocosms. Imidacloprid pulses reduced invertebrate abundance and community diversity in imidacloprid-dosed streams compared to control streams. These results correlated well with effects of imidacloprid on leaf litter decomposition and feeding rates of Pteronarcys comstocki, a stonefly, in artificial streams. Reductions in oxygen consumption of stoneflies exposed to imidacloprid were also observed in laboratory experiments. Our findings suggest that leaf litter degradation and single species responses can be sensitive ecotoxicological endpoints that can be used as early warning indicators and biomonitoring tools for pesticide contamination. The data generated illustrates the value of mesocosm experiments in environmental assessment and how the consideration of functional and structural endpoints of natural communities together with in situ single species bioassays can improve the evaluation and prediction of pesticide effects on stream ecosystems.

Laboratory toxicity test and post-exposure feeding inhibition using the giant freshwater prawn Macrobrachium rosenbergii.

A bioassay was developed using post-larvae of freshwater prawn Macrobrachium rosenbergii (length 9-10 mm) in order to determine the toxicity of profenofos, dimethoate, chlorpyrifos, carbendazim and zinc. This was the first study in the tropics with M. rosenbergii, particularly at the post-larvae stage (9-10 mm) on lethal (LC(50)) and sublethal (EC(50)) effects of toxic substances using post-exposure feeding rate as end point. Median lethal concentrations (LC(50) at 24 and 48 h) were respectively estimated as 11.6 and 9.8 microg L(-1) for profenofos, 142.1 and 102.7 microg L(-1) for dimethoate, 0.7 and 0.3 microg L(-1) for chlorpyrifos, and 439.7 and 329 microg L(-1) for zinc. Effects of carbendazim could not be estimated because carbendazim exposure needs more than 24h exposure period to produce observable effects at the concentrations used. The EC(50) using post-exposure feeding rates determined for profenofos, dimethoate, chlorpyrifos and zinc were 6.023, 269.3, 0.293 and 109.01 microg L(-1), respectively, at 24 h of exposure. Only chlorpyrifos and zinc had LC(50) concentrations greater than the post-exposure feeding EC(50) concentrations. This study demonstrated that the M. rosenbergii could also be used as a test animal to detect the effects of different chemical contaminants in aquatic environments.

Toxicity of binary mixtures of metals and pyrethroid insecticides to Daphnia magna Straus. Implications for multi-substance risks assessment.

Two different concepts, termed concentration addition (CA) and independent action (IA), describe general relationships between the effects of single substances and their corresponding mixtures allowing calculation of an expected mixture toxicity on the basis of known toxicities of the mixture components. Both concepts are limited to cases in which all substances in a mixture influence the same experimental endpoint, and are usually tested against a "fixed ratio design" where the mixture ratio is kept constant throughout the studies and the overall concentration of the mixture is systematically varied. With this design, interaction among toxic components across different mixture ratios and endpoints (i.e. lethal versus sublethal) is not assessed. In this study lethal and sublethal (feeding) responses of Daphnia magna individuals to single and binary combinations of similarly and dissimilarly acting chemicals including the metals (cadmium, copper) and the pyrethroid insecticides (lambda-cyhalothrin and deltamethrin) were assayed using a composite experimental design to test for interactions among toxic components across mixture effect levels, mixture ratios, lethal and sublethal toxic effects. To account for inter-experiment response variability, in each binary mixture toxicity assay the toxicity of the individual mixture constituents was also assessed. Model adequacy was then evaluated comparing the slopes and elevations of predicted versus observed mixture toxicity curves with those estimated for the individual components. Model predictive abilities changed across endpoints. The IA concept was able to predict accurately mixture toxicities of dissimilarly acting chemicals for lethal responses, whereas the CA concept did so in three out of four pairings for feeding response, irrespective of the chemical mode of action. Interaction effects across mixture effect levels, evidenced by crossing slopes, were only observed for the binary mixture Cd and Cu for lethal effects. The analysis of regression residuals showed that interaction effects across mixture ratios were restricted to feeding responses in binary mixtures that included Cu. These results indicate that the ability of the CA and IA concept to predict mixture toxicity effects varies from lethal to sublethal endpoints irrespective of their primary mode of action. This suggests that when considering complex responses, the pharmacological notion of mode of action should be extended to encompass an ecotoxicological mode of action, based on the concentration at which various toxicological effects become operative in the biological system under consideration.

Assessing the risks to zooplankton grazers of continuous versus pulsed cypermethrin exposures from marine cage aquaculture.

Survival responses of adult stages of the calanoid copepod A. tonsa during and after pulse exposures to cypermethrin were used to evaluate the environmental risks of cypermethrin treatments in the marine environment. To control sea lice, whole cages of salmon are exposed to cypermethrin over 1 h, after which the effluent is released into the environment and subsequently dissipates. Here we simulate this short presence of the chemical in the water column. The present study revealed that delayed toxicity in A. tonsa adults, here expressed as decreased survival, occurred following short term pulse exposures to cypermethrin. Persistent, sensitive responses to cypermethrin during and after pulse exposures of 24 h were similar to those reported in conventional acute tests, suggesting that damage caused by accumulated levels of cypermethrin during the first 24 h of exposure could explain long-term toxicity responses in A. tonsa. Delayed toxicity following 1 h pulse exposures to cypermethrin impaired male survival and therefore changed copepod sex ratios. The proposed delayed toxicity tests were considered sensitive and suggest potentially detrimental effects of cypermethrin on copepod populations within the vicinity of the cages. By monitoring toxic responses following pulsed exposures, delayed toxicity tests are especially suitable to assess environmental risks of poorly water-soluble substances that dissipate rapidly within the water column.

Genetic determination of tolerance to lethal and sublethal copper concentrations in field populations of Daphnia longispina.

In order to study the effects of environmental pollution on the genetic diversity of natural populations, two field populations of the cladoceran Daphnia longispina were sampled: one in a reference site (uncontaminated) and the other in a site historically stressed with acid mine drainage (AMD). Five hypotheses were formulated: (1) the stressed population presents a higher tolerance to lethal levels of copper than the reference population, (2) differences in tolerance to lethal levels, observed between the two populations, are due to the loss of sensitive lineages, (3) differences in tolerance to lethal levels of copper between the two populations are due to the appearance of new genotypes, (4) the acquisition of tolerance to lethal levels of copper involved changes in life-history patterns and fitness costs under optimal conditions, and (5) historical contamination by AMD resulted in tolerance differences to sublethal levels between populations, within categories similarly tolerant to lethality, specifically, lineages with similar tolerance to lethal levels from both populations show differences in tolerance to sublethal levels, the stressed population being more tolerant to sublethal levels of contamination than the reference population. Over 125 acclimated cloned lineages, from each population, were exposed to different copper concentrations for 24 hours. At the end of each assay, mortality and feeding inhibition were monitored. Life-cycle traits under optimal conditions were also monitored (time to first brood, number of neonates per brood, inter-brood time, body length, and ingested algae). At lethal levels of copper, significant differences were found in the frequency of sensitive lineages between the two populations. The stressed population did not include the most sensitive lineages, though the most tolerant ones were also present in the reference population. Thus, the hypothesis of presence of new genotypes in the stressed population resulting in an overall increase tolerance of the population at lethal levels was rejected. Changes in life-history patterns were detected, though they were not fully consistent with predictions of life-history theory. Furthermore, these changes in life-history patterns did not involve fitness costs. The fifth hypothesis was rejected since, at sublethal levels of copper, no significant differences were found between the feeding inhibitions of similarly lethal tolerant lineages of the two populations, with only one exception. Therefore, the present study further confirmed the genetic erosion hypothesis and gave further support to the incorporation of genetic diversity measurements into risk assessments.

Determining demographic effects of cypermethrin in the marine copepod Acartia tonsa: stage-specific short tests versus life-table tests.

Short-term lethal and sublethal responses of the calanoid copepod Acartia tonsa to cypermethrin were compared with life-table responses to assess whether or not it is necessary to use exposure periods longer than 5 days to estimate demographic responses to stress. More specifically, by limiting exposure periods to sensitive age classes (eggs, nauplii, copepodids, and adults) and including measurements on survival, egg production, and feeding rates, it was possible to derive a short test design of similar sensitivity and ecological relevance as full life-table tests. Short-term exposures to cypermethrin reduced copepodid's feeding rates at concentrations well below those affecting egg production rates and survival of eggs and adult stages. Lethal effects on naupliar stages occurred at lower concentrations than any other effect observed on eggs and adults. Life-table sensitivities of the intrinsic rate of increase ( r(m)) to cypermethrin were similar to those observed in short-term exposures. More specifically, exposure to cypermethrin impaired r(m) responses at concentrations (7.4 ng x L(-1)) that also affected feeding and naupliar responses. Our results show that by quantifying and separating combined toxic effects on ecologically relevant individual life-history traits, it is possible to develop toxicity test designs of similar ecological relevance yet that are less labor-intensive and costly than existing demographic tests.

Age- and sex-related variation in sensitivity to the pyrethroid cypermethrin in the marine copepod Acartia tonsa Dana.

Acute effects of cypermethrin, a pesticide used to treat ectoparasite infestations of salmon, were assessed using the calanoid copepod Acartia tonsa. A. tonsa has been widely used for hazard assessment of chemicals in the marine environment using acute toxicity tests, but only with randomly selected adults, assuming a sex ratio of 1:1. The present study assesses the environmental hazard of cypermethrin by exposing nauplii and adult males and females, separately. Our results showed that the naupliar stages were 28 times more sensitive to cypermethrin than adults after 96 h of exposure, with LC50s of 0.005 microg x L(-1) and 0.142 microg x L(-1), respectively. Significant differences in sensitivity between sexes were only found during the first 24 h of exposure, with males being approximately twice as sensitive as females. The results of age-related variation in sensitivity are supported by studies with other species of copepods and toxicants, where the increased capacity of the adults for detoxification, the allometric differences in weight and size, and the molting process are given as explanations. Variation in sensitivity between sexes is discussed in terms of faster depuration rates in females through egg production and implications of feeding rate changes after 24 h of exposure. Our results suggested that standard toxicity test methods using A. tonsa are unsatisfactory because the most sensitive life stage is not included and sex-related differences in tolerance are not taken into account. We also found that cypermethrin caused significant mortality in Acartia at exposures concentrations from one to three orders of magnitudes lower than the recommended field treatment concentration for copepodic infestations.

Biochemical factors contributing to response variation among resistant and sensitive clones of Daphnia magna Ssraus exposed to ethyl parathion.

Biochemical factors contributing to response variation in five clones of Daphnia magna exposed to ethyl parathion were studied. Differences in sensitivities to ethyl parathion between sensitive and resistant clones varied between four- and ninefold. Acute toxicity and in vivo acetylcholinesterase (AChE) inhibition responses to ethyl parathion were similar, whereas in vitro AChE sensitivities to paraoxon were higher. Thus indicating that, in vivo, not all ethyl parathion present is converted by Daphnia juveniles to active metabolite, or if so, only a proportion of the paraoxon produced is in direct contact with its target. Comparison of AChE activities obtained during in vitro and in vivo exposures with acute responses revealed clonal concordance only between in vivo AChE sensitivities and acute toxicity. This latter finding indicates that clonal differences in ethyl parathion metabolism rather than differences in AChE sensitivities are the most likely explanations of the observed genetic differences in tolerance.

Comparing metal toxicity among Daphnia magna clones: an approach using concentration-time-response surfaces.

This study investigates the use of concentration-time-response surfaces as a tool to predict potential long-term effects of metals to Daphnia magna, using mortality as an endpoint. Specifically, concentration-time-response surfaces were determined for four D. magna clones exposed to four metals (Cd, Zn, Cu or U) in moderate-hard or hard synthetic freshwater for 24-96 h. Mortality data were log(e) transformed into probits and then regressed against the reciprocal of exposure time and concentration. The results obtained clearly showed that mortality was markedly affected at exposure periods longer than 48 h. Thus, an optimal exposure period of 72 h was selected to compare predicted lethality effects (LC) of different intensities (i.e., 10-50%) derived from concentration-time-response surfaces with measured chronic lethal levels obtained from the literature. Only the results of Cd and Cu show good agreement between predicted and measured chronic lethal concentration levels. The apparent disagreement observed for U could be attributed to differences in water quality, and hence, U bioavailability. The high levels of Zn toxicity observed in relation to the predicted values could indicate that chronic mortality of Zn is mediated by toxic anorexia. Overall, the results obtained show that concentration-time-response surfaces offered the potential to assess the effect of time on toxicity, which is desirable to credibly extrapolate from acute to chronic scenarios. Furthermore, by determining lower mortality thresholds (i.e., LC(10)) at different times, concentration-time-response surfaces were able to emulate the mode of action of the selected metals on chronic exposures. Therefore, the use of concentration-time-response surfaces has potential application in environmental risk assessment.

An early life-stage test with Daphnia magna Straus: an alternative to the 21-day chronic test?

Chronic life-cycle tests using the freshwater cladoceran Daphnia magna aim to measure the effect of toxic chemicals on adult reproduction, in terms of the number of viable offspring produced. A tacit assumption is made that the primary effect of toxicants in these tests is a sublethal effect on the parent, reducing egg production and hence fecundity. The authors' observations on two chemicals, sodium bromide and 3,4-dichloroaniline, demonstrate that this assumption is at least partly false. In these experiments, the primary toxic effect of these compounds was to kill developing eggs in the brood chamber. Total egg output was unaffected, whereas output of viable eggs was severely impaired--an acute lethal effect. A further experiment demonstrated that the effect was limited to those eggs developing in the brood chamber, with eggs in the ovary remaining unaffected. It was suggested that these observations may provide the basis for the development of a short, sensitive early life-stage test with this species.

A comparative study of genotype sensitivity to acute toxic stress using clones of Daphnia magna straus.

The genetic typing of laboratory strains of Daphnia magna revealed that although there are genetic differences between different testing laboratory strains, most laboratories culture single genotypes as clones. A comparative study was made of the performance of five of the more common clones in terms of neonate mortality under acute exposure to nine toxic chemicals. The range in clonal EC50 response was from less than one to more than two orders of magnitude in terms of concentration. Surprisingly, there was little pattern in rank order of response of clones exposed to different toxicants, thus suggesting that genotype-environment interactions were predominating. Nevertheless, it was suggested that specifying the genotype used in D. magna toxicity tests will result in increased test repeatability. Evidence indicating that culture environment may also significantly affect the performance of neonates in acute toxicity tests was also presented.