Toxicity of Binary Metal Mixtures to the Tropical Ostracod Strandesia Trispinosa.

The ecotoxicity of metals is generally assessed individually, in part because current knowledge does not allow for the accurate prediction of the toxicity of metal mixtures to aquatic organisms. The objective of this study was to investigate the toxic effects of binary combinations of metal salts (copper sulphate-CuSO4, cadmium chloride-CdCl2, mercury chloride-HgCl2 and manganese sulphate-MnSO4) on the tropical ostracod Strandesia trispinosa through acute toxicity tests. To this end, ostracods were exposed to each individual metal salt as well as to their combinations by applying a full factorial design. The model that best explained the effects of the mixtures CuSO4 x CdCl2, CuSO4 x HgCl2 and CuSO4 x MnSO4 on the survival of S. trispinosa was Concentration Addition, whereas this was Independent Action for the CdCl2 x HgCl2 mixture. The observed synergistic interactions are likely to result in unacceptable risks to aquatic ecosystems under real field conditions. This is especially the case if CuSO4 predominates the metal mixture, as observed for its combination with mercury and manganese.

Acute toxicity of four metals to three tropical aquatic invertebrates: The dragonfly Tramea cophysa and the ostracods Chlamydotheca sp. and Strandesia trispinosa.

The relatively low availability of toxicity data for indigenous tropical species has often been discussed. In addition, several taxonomic groups of invertebrates are understudied, such as dragonflies and ostracods. The aim of the present study was therefore to evaluate the acute toxicity of four metals (cadmium - Cd, copper - Cu, manganese - Mn, and mercury - Hg) to the tropical dragonfly nymphs of Tramea cophysa and two tropical ostracod species (Chlamydotheca sp. and Strandesia trispinosa). Toxicity data for other invertebrates were also mined to allow comparing the sensitivity of the three test species with that of other (temperate and tropical) invertebrates. The order of metal sensitivity was different for the three test species: T. cophysa: Cu > CdHg > Mn, Chlamydotheca sp.: Cd > Cu > Hg > Mn, and S. trispinosa: Cd > Hg > Cu > Mn. However, manganese was the least toxic metal tested for all three species, which is hypothesized to be due to a possible metal transfer to the cuticle of the moulting test species. The sensitivity ranking of the three test species to the metals was S. trispinosa > Chlamydotheca sp.>T. cophysa (except for Cu for which the ranking was Chlamydotheca sp.>T. cophysa > S. trispinosa). Overall, the test species are concluded to be suitable test organisms for tropical toxicity evaluations. Future studies should also evaluate the chronic toxicity and include other important metal exposure routes such as sediment and food.

Effects of florfenicol and oxytetracycline on the tropical cladoceran Ceriodaphnia silvestrii: A mixture toxicity approach to predict the potential risks of antimicrobials for zooplankton.

Antimicrobials are commonly used in aquaculture to treat infectious diseases in fish. The overuse of these chemicals, however, has made them a contamination source for the aquatic environments. In this study, single and combined effects of florfenicol (FLO) and oxytetracycline (OTC), two antimicrobials widely used in the fish farming, were evaluated in acute and chronic toxicity tests using the tropical cladoceran Ceriodaphnia silvestrii as a model species. Also, a preliminary risk characterization of FLO and OTC for zooplankton was carried out, taking into account different exposure scenarios. The results obtained revealed that FLO and OTC have adverse effects on the mobility, reproduction and population growth rate of C. silvestrii in single exposures. In addition, mixture effects on the C. silvestrii were more severe than predicted effects based on the Concentration Addition model, showing a synergistic deviation for the mobility and a dose-level dependent deviation for the reproduction (synergism at higher levels than EC60). In relation to the risk characterization, risk quotients (RQs) exceeded 1 for chronic toxicity data obtained in both OTC and mixture exposures, indicating that the concentrations of these chemicals in Brazilian freshwater bodies could potentially present risks for the reproduction of zooplankton species in tropical regions. The RQs obtained for the mixtures were higher than those obtained for each chemical separately. Therefore, it is highly recommended that RQs are derived from single and mixture exposure data in order to obtain a more accurate risk characterization.

Lethal and sublethal toxicity of abamectin and difenoconazole (individually and in mixture) to early life stages of zebrafish.

In recent years, the need for the development of alternative test methods for the conventional acute fish toxicity test (AFT) with adult fish has often been discussed. In addition, concerns have been raised on the potential risks related with environmentally realistic pesticide mixtures since risk evaluations have traditionally been based on individual pesticides. The insecticide/acaricide abamectin and the fungicide difenoconazole are the main pesticides that are intensively used in Brazilian strawberry crop and are hence likely to occur simultaneously in edge-of-field waterbodies. The aim of the present study was therefore to evaluate the lethal and sublethal toxicity of single and mixture exposures of these pesticides to zebrafish early life stages (embryos and juveniles). By comparing the derived toxicity data of the individual compounds with that previously determined for zebrafish adults, the order of life stage sensitivity was juvenile > adult > embryo. The pesticide mixture revealed a dose-level dependent deviation of the independent action model, with antagonism at low dose levels and synergism at high dose levels. Sublethal parameters (especially those related with locomotion) were considerably more sensitive than lethality. Subsequently, the inclusion of sublethal parameters may greatly improve the sensitivity of FET tests and hence its suitability as a substitution of adult fish testing in risk assessment evaluations.

Sensitivities of three tropical indigenous freshwater invertebrates to single and mixture exposures of diuron and carbofuran and their commercial formulations.

As compared to their temperate counterparts, few toxicity tests have been conducted so far into the evaluation of the sensitivity of indigenous tropical species to pesticides. Especially mixture toxicity assessments appear to be scarce. To contribute to increase our knowledge in this arena, we evaluated the acute toxicity of diuron and carbofuran and their mixtures to the neotropical oligochaetes Allonais inaequalis and Dero furcatus, and the ostracod Strandesia trispinosa. Tests were performed with both the pure active ingredients, as well as their formulated products. The toxicity of the latter to the three test organisms was generally greater than that of the pure active ingredients, although absolute differences were rather small. The sensitivity of the indigenous species was slightly greater than temperate test species from the same taxonomic groups. The concentration addition conceptual model best described the results of the mixture toxicity data. Derived deviations of this model appeared to be dependent on the test organism and as to whether the pesticides were applied as active ingredients or their commercial products. Reported field concentrations of the two pesticides indicate risks to freshwater biota, especially if they are both present. The test species used in the present study are concluded to be suitable candidates as surrogate test organisms in local pesticide risk evaluations.

Single and mixture toxicity of abamectin and difenoconazole to adult zebrafish (Danio rerio).

Concerns have been raised in recent years on the potential risks related with pesticide mixtures that are likely to be present in agricultural edge-of-field waterbodies. Despite the high use of pesticides in tropical countries like Brazil, studies evaluating pesticide mixtures are especially scarce in the tropics. The insecticide abamectin and the fungicide difenoconazole are the main pesticides intensively used in Brazilian strawberry crop and are hence likely to occur simultaneously. The aim of the present study was therefore to evaluate the toxicity of abamectin, difenoconazole and their mixture to the tropical fish Danio rerio. Laboratory toxicity tests with the individual pesticides indicated 48 h-LC50 values of 59 µg L-1 for abamectin and 1.4 mg L-1 for difenoconazole. Mixtures of the two pesticides revealed a synergistic deviation of the independent action model. Implications of study findings for the aquatic risk assessment of pesticide mixtures, especially in tropical countries and indications for future research are discussed.

Toxicity of abamectin and difenoconazole mixtures to a Neotropical cladoceran after simulated run-off and spray drift exposure.

Aquatic risk assessments of pesticides in tropical countries have often been disputed for being largely based on risk evaluations conducted in temperate regions. Although pesticide sensitivity comparisons between temperate and tropical freshwater organisms have indeed not revealed consistent differences, risk assessments are currently still based on a relatively small tropical toxicity dataset. In addition, greater levels of runoff and spray drift may be expected in tropical than in temperate agroecosystems, indicating that aquatic life in edge-of-field water bodies is likely to be subjected to higher concentrations of pesticides and their mixtures. The aim of the present study was to evaluate the toxicity of Kraft® 36 EC (a.i. abamectin), Score® 250 EC (a.i. difenoconazole) and their mixture to the Neotropical cladoceran Macrothrix flabelligera. Laboratory toxicity tests with the individual formulated products indicated EC50-48h values of 3.1 and 659µg a.i./L given as nominal test concentrations, respectively. Mixtures of the two pesticides revealed a concentration-dependent deviation of the independent action model, with antagonism at low and synergism at high pesticide mixture concentrations. Laboratory toxicity tests were also conducted with microcosm water that was treated with the individual or mixtures through runoff or direct overspray. Microcosm tanks receiving runoff water from experimental soil plots applied with recommended doses of the individual pesticides did not show toxicity to the test organism. Microcosms that received runoff water containing the pesticide mixture, however, did cause a short-term effect on immobility. The microcosms that were treated by direct overspray of both pesticide formulations showed the most pronounced toxic effects. Study findings suggest a potential risk of these pesticides at environmentally relevant concentrations, especially when they are both present.

Use of cholinesterase activity as an ecotoxicological marker to assess anatoxin-a(s) exposure: Responses of two cladoceran species belonging to contrasting geographical regions.

The specificity of cholinesterase (ChE) activity to detect the presence of anatoxin-a(s) and sublethal effects of a 7-day exposure to Anabaena spiroides extract containing anatoxin-a(s) were assessed in two freshwater cladoceran species. Activities of ChE of both Pseudosida ramosa and Daphnia magna can be used to indicate the presence of the neurotoxin anatoxin-a(s), but not for the hepatotoxic microcystin. Activity of ChE of P. ramosa, however, performed better as a biomarker of exposure to A. spiroides than that of D. magna. Furthermore, sublethal exposure to A. spiroides extract significantly inhibited the ChE activity in P. ramosa and negatively affected both individual and population endpoints. For D. magna, the inhibition of ChE activity was not related to effects at higher levels of biological organization, since no direct effect was recorded on the individual and population endpoints. The activity of ChE in P. ramosa also proved to be a good predictor of chronic effects of the A. spiroides extract at higher levels of biological organization, since 48-h ChE inhibition was linked to the sublethal effects on the individual and population. These relationships could not be established for D. magna. Since relationships between the effects of A. spiroides extract at different levels of biological organization were species-specific, it can be concluded that the choice of test organism interferes with the accuracy of the environment risk assessment of this neurotoxin and, hence, the use of native species is recommended for its assessment.

Measurements of cholinesterase activity in the tropical freshwater cladoceran Pseudosida ramosa and its standardization as a biomarker.

The activity of cholinesterases (ChE) has been recognized as a useful tool for assessing the toxicity in the environmental assessment programs. Nevertheless, the prior optimization of the experimental conditions for the appropriate measuring of the ChE activity enables us to get reliable results. Thus, the main objective of this study was to adapt and optimize a microplate assay for measuring the activity of ChE in the tropical cladoceran Pseudosida ramosa. The best readings for the reaction rates were obtained with buffers of pH 8.0 and molarity of 0.02M. The measurements of the reaction rates for the different substrate concentrations showed that the maximum reaction rate (32mODmin(-1)) was achieved by the final concentration of 2mM of substrate. In relation to the enzyme concentration, reaction rates were directly proportional to the protein concentration, which confirmed the linear kinetics for a maximum reaction rate. On the basis of the results of the assays for the effect of the number of individuals and homogenate dilution on the reaction rate of substrate hydrolysis and ChE activity, we recommend using of 30 individuals (3 days-old) in 250µL of buffer, 20 individuals (7 days-old) in 250µL of buffer and 15 individuals (both 14 and 21 days-old) in 300µL of buffer. The limits of quantitation obtained were 1.419mODmin(-1) (≤72h-old), 1.670mODmin(-1) (7 days-old), 0.943mODmin(-1) (14 days-old) and 0.797mODmin(-1) (21 days-old). In conclusion, it was possible to measure the ChE activity in P. ramosa with the methodology adapted, thus contributing to the implementation of a biochemical biomarker in freshwater toxicity assessments in tropical regions.

Acute effects of Anabaena spiroides extract and paraoxon-methyl on freshwater cladocerans from tropical and temperate regions: links between the ChE activity and survival and its implications for tropical ecotoxicological studies.

Cholinesterase (ChE) activity was measured in Pseudosida ramosa and Daphnia magna, which had previously been exposed to Anabaena spiroides extract or to paraoxon-methyl for 48 h. These activities were then related to survival at 48 h. For A. spiroides extract, the observed 48-h LC50 was 2.27 and 2.70 × 10(6)cells mL(-1), while for paraoxon-methyl it was 0.60 and 2.17 µg L(-1), respectively, for P. ramosa and D. magna. Dose-response relationships were obtained for both P. ramosa and D. magna, when exposed to A. spiroides extract or paraoxon-methyl. Thus, when the tested concentrations of the toxicants increased, ChE activity and survival decreased. The ratio between 48-h IC50 for ChE and 48-h LC50 ranged from 75% to 81% for P. ramosa and from 77% to 81% for D. magna. This indicated that the concentrations of both A. spiroides extract and paraoxon-methyl that cause 50% mortality also inhibit ChE activity by 50%. Also, it was found that, for P. ramosa, a 50% inhibition of ChE activity was associated with a survival of 59.5% and 60.9%, respectively, for A. spiroides extract and paraoxon-methyl. However, for D. magna, at high levels of inhibition of ChE activity, almost no mortality was detected. In this specific case, 50% inhibition of the ChE activity was associated with 90.4 and 95.4% survival for A. spiroides extract and paraoxon-methyl, respectively. In contrast, enzyme inhibition slightly above 60% had a strong detrimental effect on survival in D. magna. These different patterns found in the relationship between ChE inhibition and survival may be due to species-specific differences in the affinities of both acetylcholinesterase and pseudocholinesterases, since the cladoceran ChE assays were performed with whole-body homogenates. In conclusion, when using ChE as a biochemical biomarker in risk assessment of cyanobacterial neurotoxic blooms in tropical regions, it is strongly recommended that native species are used, since our results revealed that P. ramosa was more sensitive than D. magna for both assay endpoints and both toxicants. Furthermore, the relationship between ChE activity and survival had a species-specific response. Therefore, the use of the model species D. magna in acute toxicity tests and ChE assays in tropical regions may lead to errors in the estimation of risks to the local species.

Can mixtures of cyanotoxins represent a risk to the zooplankton? The case study of Daphnia magna Straus exposed to hepatotoxic and neurotoxic cyanobacterial extracts.

Worldwide, cyanobacterial blooms have been increasing in intensity and frequency, with toxic cyanobacteria sometimes dominant throughout the year in many freshwater bodies. Since the coexistence of more than one type of cyanotoxins in freshwater environments is a common phenomenon, studies on the joint effects of these toxins would be very useful. In this study, the single and combined effects of two cyanotoxins with different modes of action (hepatotoxic and neurotoxic) on the survival (lethal exposure) and feeding (sublethal exposure) of the cladoceran Daphnia magna were investigated. With the single exposures, it was observed that both the survival and feeding activity of the daphnids were impaired by the hepatotoxic and neurotoxic extracts at environmentally relevant concentrations. In the combined exposures, both survival and feeding rate endpoints showed a good fit to the independent action model. For the acute assay and 24h exposure period in the feeding inhibition test, there was no interaction between components of the hepatotoxic and neurotoxic extracts, although a slight tendency to a synergistic deviation could be seen in the feeding rates. On the other hand, for the 4h post-exposure period, a synergistic deviation was found in feeding rates at all mixture concentrations tested. Hence, the combined exposure of hepatotoxins and neurotoxins should also be taken into account in risk assessments of freshwater bodies, since the mixture of these toxins can result in more severe post-exposure effects on the feeding of daphnids than the sum of those expected for single exposures.

Acute and chronic toxicity of chromium and cadmium to the tropical cladoceran pseudosida ramosa and the implications for ecotoxicological studies.

In this study, the acute and chronic toxicity of the metals chromium and cadmium were tested against the tropical freshwater cladoceran Pseudosida ramosa. Acute tests showed that the 48-h LC50 of chromium was 29 µg L(-1) , while that of cadmium was 12 µg L(-1) . P. ramosa had a similar sensitivity to those of other cladoceran species cultured in the same conditions as in this study, or a higher sensitivity when water hardness was raised. Long-term chronic exposure of P. ramosa to chromium decreased maternal survival, fecundity, and fertility at a concentration of 10 µg L(-1) . For cadmium, reductions in the same endpoints were observed at a concentration of 3 µg L(-1) . Moreover, the degree of toxicity of the chromium and cadmium to P. ramosa after release of the first brood was similar to the result obtained after 21 days, using reproduction as the endpoint. Brazilian water bodies located near industrial areas already show concentrations of chromium and cadmium higher than levels causing acute and chronic toxicity to P. ramosa in our study. Many water quality criteria in tropical regions are based on ecotoxicological tests with non-native species and, consequently, this may lead to errors of interpretation when the permitted maximum levels of for each toxic substance are established. Therefore, we reinforce the idea of using native species to establish the maximum concentrations of toxic substances in water quality criteria, especially for metals, since their effects are related to water hardness, pH, and temperature specific to each region.

Acute and chronic effects of atrazine and sodium dodecyl sulfate on the tropical freshwater cladoceran Pseudosida ramosa.

Toxicities of atrazine and sodium dodecyl sulfate (SDS) to the tropical freshwater cladoceran Pseudosida ramosa were studied in the laboratory. Acute tests showed that the 48-h LC50 of atrazine was 20.9 mg l⁻¹, while that of SDS was 11.1 mg l⁻¹. P. ramosa showed to be slightly more sensitive than the other species of temperate cladocerans, in the assay conditions specified for each one. Long-term exposure of P. ramosa individuals to atrazine decreased the 21-day fecundity, the 21-day fertility and r(m), at concentrations ranging from 0.8 to 3.2 mg l⁻¹. Furthermore, fecundity and fertility at each brood decreased from the first to the fifth, at concentrations ranging from 0.8 to 3.2 mg l⁻¹ and for the first three broods at the concentration of 0.4 mg l⁻¹. Long-term exposure of female P. ramosa to SDS decreased the 21-day fecundity, the 21-day fertility and r(m), at concentrations of 2 and 4 mg l⁻¹. Fecundity and fertility of each brood were reduced from the first to the fifth, at concentrations of 2-4 mg l⁻¹, and for the first three at concentrations of 0.5 and 1 mg l⁻¹. The survival and moulting of the adult females were not affected by either chemical at the concentrations tested. Many water quality criteria in tropical regions are based on ecotoxicological tests with non-native species and this may lead to errors in setting the maximum permissible levels of chemicals in water bodies. Therefore, we reiterate here the idea of using native species in ecotoxicological assessments.

Acute and chronic effects of sodium and potassium on the tropical freshwater cladoceran Pseudosida ramosa.

In this study, the toxicities of sodium and potassium to the tropical freshwater cladoceran Pseudosida ramosa were assessed. Acute toxicity tests on this species showed that the 48-h LC(50) of Na(+) was 556 mg l(-1), while that of K(+) was 17.7 mg l(-1). Long-term exposure of female P. ramosa to sodium reduced the total number of survivors from 10 to 6 at a concentration of 249 mg l(-1), 21-day fecundity from 20.4 to 14.3 eggs female(-1) at concentrations ranging from 72 to 249 mg l(-1), 21-day fertility from 20.1 to 6.5 neonates female(-1) at concentrations ranging from 25 to 249 mg l(-1). Furthermore, fecundity of each brood from the second to the fifth was significantly lower at 249 mg l(-1) and fertility of each brood from the first to the fifth at concentrations ranging from 25 to 249 mg l(-1). A significant decrease in fertility was associated with an increase in the number of aborted eggs. Long-term exposure to potassium decreased the 21-day fecundity of P. ramosa from 14.2 to 10.8 eggs female(-1) at a concentration of 11 mg l(-1) and fertility (fourth brood only) at 6.2 and 11 mg l(-1). Tropical reservoirs located near areas where the soil is overloaded with fertilizers and ferti-irrigation with vinasse already show concentrations of Na(+) and K(+) very close to those producing sub-lethal long-term effects on P. ramosa. A possible consequence is that organisms of the aquatic biota cannot adapt and freshwater taxa may become locally extinct, transferring dominance to salt-tolerant taxa.

Acute toxicity tests with the tropical cladoceran Pseudosida ramosa: The importance of using native species as test organisms.

Cladocerans have long been used for toxicological assessments of a diverse range of substances. The use of cladocerans in toxicity tests has many advantages, such as their short life cycle, parthenogenetic reproduction (clones), and high sensitivity to toxicants, as well as the easy laboratory maintenance of cultures. The most commonly used cladoceran in ecotoxicological studies of aquatic environments is undoubtedly Daphnia magna. Standard methods using cladocerans as test organisms have been documented and adopted by major international organizations and regulatory agencies of many countries. However, today there is a growing need for improving test organisms and protocols to better reflect local species sensitivity or site-specific conditions. The present study aimed to assess the tropical species Pseudosida ramosa as a potential test organism for ecotoxicological purposes, by carrying out standard acute tests with six reference compounds. Based on the results obtained in the present study and in comparison with other cladocerans, it was found that P. ramosa was more sensitive than Daphnia magna, had a sensitivity similar to that of Daphnia similis, and was less sensitive compared to Ceriodaphnia dubia and C. silvestrii (Neotropical species), except for the salts, sodium chloride and potassium chloride. Also, when P. ramosa was compared with test organisms of other taxonomic groups, we observed that it was more sensitive than most of the others, from simple coelenterates to complex fish. Considering these results and the wide distribution of the cladoceran P. ramosa in tropical and subtropical regions, we suggest that this species can be adopted as a test organism, being a good substitute for the exotic daphnid D. magna, for monitoring of toxicants in freshwaters.