An alternative approach to assess ecotoxicological effects of agrochemical combinations used in Brazilian aquaculture farms.

Agrochemicals used for treating and preventing aquaculture diseases are usually present in combination with other compounds, and the toxicity resulting from their chemical interactions presents an important reason to assess the ecotoxicity of compound mixtures in view to better understanding the joint action of chemicals and avoiding their environmental impacts. In this study, we evaluated the acute aquatic ecotoxicity of several compounds used in Brazilian fish farming (Oxytetracycline [OXT], Trichlorfon [TRC], and BioFish® [BIO]), both individually and in binary and ternary mixtures. Initial test concentrations were prepared according to the recommended concentrations for aquaculture application, and from these, a geometric dilution series was tested on two important fresh water quality indicator species, the microcrustacean Daphnia magna and the bacterium Aliivibrio fischeri. At the recommended pond application rate, TRC and BIO applied individually showed toxicity to the tested organisms in terms of the lowest-observed-effect concentration (LOEC), and D. magna was always more sensitive than A. fischeri. For the two test organisms, the results obtained with the binary mixtures showed that the TRC and BIO mixture was more toxic than TRC and OXT, which in turn was more toxic than OXT and BIO. The toxicity from all agrochemicals in the ternary mixture was more than that of the agrochemical combinations in the binary mixtures. Given the results presented in this study, it is evident that the mode of action and availability of the tested compounds undergo changes that increase toxicity when they are present in combination, and therefore, aquaculture wastewater treatment should be adopted to ensure decontamination of agrochemical residues.

A 3D ecotoxi-topological profile: Using concentration-time-response surfaces to show peroxidase activity in Zea mays (L.) exposed to aluminium or arsenic in hydroponic conditions.

This study sought to use concentration-time-response surfaces to show the effects of exposure to toxic (semi-)metals on peroxidase activity in higher plants as a function of exposure-concentration and exposure-time. Maize (Zea mays L.) seedlings (i.e., leaves and roots) were exposed to arsenic (as As3+) or aluminium (as Al3+) under hydroponic conditions, and their biomass and peroxidase enzyme responses were assessed at different concentration-time-exposures. The 3D ecotoxi-profile generated with these data showed two distinct regions: the first region is formed by exposures (i.e., points for time-concentration pairings) that were not statistically different from the results of the control points (i.e., zero toxicant concentration and all exposure-times), whereas the second region is formed by exposure pairings with results that were statistically different to those obtained from control pairings. Overall, the data show that enzyme activity increased over a shorter exposure-time when there was an increase in the exposure-concentration of the toxicant, which can be seen on a 3-D toxicity profile. We propose that quantitative relationship ratios from different assessed endpoints (e.g., biomass and enzyme activity) and enzymatic concentration-time-response surfaces could be helpful in the field of environmental-policy management.

Microplastics enhance Daphnia magna sensitivity to the pyrethroid insecticide deltamethrin: Effects on life history traits.

The aim of the study was to investigate the influence of microplastics (MPs) on the ecotoxicity of common contaminants of aquatic ecosystems. As a model contaminant, the hydrophobic pesticide deltamethrin (DM) was chosen, and its effects on life history traits of Daphnia magna were studied in the presence or absence of polyethylene MPs. Commercialized DM and MPs obtained as dry powder were used in the experiment. According the manufacturer (Cospheric, Santa Barbara, CA, USA) MPs were spherical (1-4 µm in diameter), had a density of 0.96 g/cm-3 and were without any solvent. Three concentrations of polyethylene MPs were tested (0, 1, 10 mg/L) with two realistic concentrations of DM (0 and 40 ng/L) and a solvent control (acetone). During the 21 d experiment, D. magna neonates were individually exposed to the treatments, and the effects of MPs and DM alone and together were evaluated by assessing survival, number of cumulative molts, days to first brood, number of broods, number of neonates per surviving adult, and body length. Significant detrimental effects on survival were only observed for the two mixture treatments. DM alone (40 ng/L) delayed the days to first brood and reduced the number of neonates per surviving adult, whereas MPs alone (10 mg/L) induced significant reduction in the number of juveniles by surviving adults. The combined exposure to DM and MPs clearly had a synergistic effect on survival, brood number, and number of neonates per surviving female. For example, compared to exposure to 40 ng/L of DM alone, the addition of 1 mg/L of MPs resulted in a 51.1% reduction in number of neonates per surviving female and a 46% reduction in brood number. These results suggest the potential drastic effects of this kind of mixed exposure on daphnid populations, which are key components of freshwater food webs.

Microplastics in freshwater environment: the first evaluation in sediments from seven water streams surrounding the lagoon of Bizerte (Northern Tunisia).

Microplastic (MP) concentrations were determined, for the first time, in surface sediment of seven streams around the lagoon of Bizerte (Northern Tunisia), using a saturated NaCl flotation technique. Microplastics were categorised according to type, colour and size using a stereoscopic microscope. Results showed that all sediment samples contained MPs. The greatest MP abundance was observed at Jedara stream (6920 ± 395.98 items kg-1 dry weight), while the lowest mean value was 2340 ± 227.15 items kg-1 dry weight at Khima stream. The highest numbers of MPs were from streams near populated areas and municipal and industrial effluent discharges. Samples were made up entirely of secondary MPs mainly fibres, followed by fragments and films. The predominant colours were as follows: black > clear > white > red > blue > green > yellow for fibres, white > blue > black > red for fragments and red > white > clear > green > blue = black for films. Microplastic particles in the samples ranged from 0.2 to 5 mm in length. FTIR analysis revealed that the abundant polymers were polypropylene and polyethylene. This work contributes to the growing evidence that MP contamination is widespread even in freshwater ecosystems and provides a baseline for future studies and risk assessments.

Combined acute ecotoxicity of malathion and deltamethrin to Daphnia magna (Crustacea, Cladocera): comparison of different data analysis approaches.

We studied the combined acute effect (i.e., after 48 h) of deltamethrin (a pyrethroid insecticide) and malathion (an organophosphate insecticide) on Daphnia magna. Two approaches were used to examine the potential interaction effects of eight mixtures of deltamethrin and malathion: (i) calculation of mixture toxicity index (MTI) and safety factor index (SFI) and (ii) response surface methodology coupled with isobole-based statistical model (using generalized linear model). According to the calculation of MTI and SFI, one tested mixture was found additive while the two other tested mixtures were found no additive (MTI) or antagonistic (SFI), but these differences between index responses are only due to differences in terminology related to these two indexes. Through the surface response approach and isobologram analysis, we concluded that there was a significant antagonistic effect of the binary mixtures of deltamethrin and malathion that occurs on D. magna immobilization, after 48 h of exposure. Index approaches and surface response approach with isobologram analysis are complementary. Calculation of mixture toxicity index and safety factor index allows identifying punctually the type of interaction for several tested mixtures, while the surface response approach with isobologram analysis integrates all the data providing a global outcome about the type of interactive effect. Only the surface response approach and isobologram analysis allowed the statistical assessment of the ecotoxicological interaction. Nevertheless, we recommend the use of both approaches (i) to identify the combined effects of contaminants and (ii) to improve risk assessment and environmental management.

Use of phytoproductivity data in the choice of native plant species to restore a degraded coal mining site amended with a stabilized industrial organic sludge.

Coal mining-related activities result in a degraded landscape and sites associated with large amounts of dumped waste material. The arid soil resulting from acid mine drainage affects terrestrial and aquatic ecosystems, and thus, site remediation programs must be implemented to mitigate this sequential deleterious processes. A low-cost alternative material to counterbalance the affected physico-chemical-microbiological aspects of the degraded soil is the amendment with low contaminated and stabilized industrial organic sludge. The content of nutrients P and N, together with stabilized organic matter, makes this material an excellent fertilizer and soil conditioner, fostering biota colonization and succession in the degraded site. However, choice of native plant species to restore a degraded site must be guided by some minimal criteria, such as plant survival/adaptation and plant biomass productivity. Thus, in this 3-month study under environmental conditions, phytoproductivity tests with five native plant species (Surinam cherry Eugenia uniflora L., C. myrianthum-Citharexylum myrianthum, Inga-Inga spp., Brazilian peppertree Schinus terebinthifolius, and Sour cherry Prunus cerasus) were performed to assess these criteria, and additional biochemical parameters were measured in plant tissues (i.e., protein content and peroxidase activity) exposed to different soil/sludge mixture proportions. The results show that three native plants were more adequate to restore vegetation on degraded sites: Surinam cherry, C. myrianthum, and Brazilian peppertree. Thus, this study demonstrates that phytoproductivity tests associated with biochemical endpoint measurements can help in the choice of native plant species, as well as aiding in the choice of the most appropriate soil/stabilized sludge proportion in order to optimize biomass production.

Effect of carbaryl (carbamate insecticide) on acetylcholinesterase activity of two strains of Daphnia magna (Crustacea, Cladocera).

The present study was designed to investigate the effect of carbaryl (carbamate insecticide) on the acetylcholinesterase activity in two strains (same clone A) of the crustacean cladoceran Daphnia magna. Four carbaryl concentrations (0.4, 0.9, 1.8 and 3.7 µg L(-1)) were compared against control AChE activity. Our results showed that after 48 h of carbaryl exposure, all treatments induced a significant decrease of AChE activities whatever the two considered strains. However, different responses were registered in terms of lowest observed effect concentrations (LOEC: 0.4 µg L(-1) for strain 1 and 0.9 µg L(-1) for strains 2) revealing differences in sensitivity among the two tested strains of D. magna. These results suggest that after carbaryl exposure, the AChE activity responses can be also used as a biomarker of susceptibility. Moreover, our results show that strain1 is less sensitive than strain 2 in terms of IC50-48 h of AChE activity. Comparing the EC50-48 h of standard ecotoxicity test and IC50-48 h of AChE inhibition, there is the same order of sensitivity with both strains.

Use of agro-industrial organic sludge amendment to remediate degraded soil: chemical and eco(geno)toxicological differences between fresh and stabilized sludge and establishment of application rates.

Soil degraded by coal mining activities can be remediated by amendment with agro-industrial organic sludge. However, the environmental impacts associated with this management practice must be properly addressed. In this context, the objective of this study was to evaluate the eco(geno)toxicity of a fresh and a stabilized sludge before use in a laboratory soil remediation test. Chemical analysis of the complex mixtures (degraded soil, fresh sludge, and stabilized sludge) was carried out, as well as a battery of eco(geno)toxicity tests on microbiological enzymes (fluorescein hydrolysis), earthworms, and higher plants (including Vicia faba genotoxicity test), according to published methodologies. The results of these tests showed that fresh sludge was more toxic than sludge stabilized over 6 months toward earthworms and higher plants (lettuce, corn, and wild cabbage), while phyto(geno)toxicity tests with V. faba indicated the same genotoxicity levels for the two types of sludge. In the soil remediation simulation using different mixtures of degraded soil and stabilized sludge, the proportions of 50:50% (dry weight basis) provided the lowest phyto(geno)toxicity effects and this mixture can be used for the revegetation of the contaminated site.

Acute and chronic ecotoxicity of carbaryl with a battery of aquatic bioassays.

The ecotoxic effects of carbaryl (carbamate insecticide) were investigated with a battery of four aquatic bioassays. The nominal effective concentrations immobilizing 50% of Daphnia magna (EC50) after 24 and 48 h were 12.76 and 7.47 µg L(-1), respectively. After 21 days of exposure of D. magna, LOECs (lowest observed effect concentrations) for cumulative molts and the number of neonates per surviving adult were observed at carbaryl concentration of 0.4 µg L(-1). An increase of embryo deformities (curved or unextended shell spines) was observed at 1.8 and 3.7 µg L(-1), revealing that carbaryl could act as an endocrine disruptor in D. magna. Other bioassays of the tested battery were less sensitive: the IC50-72h and IC10-72h of the algae Pseudokirchneriella subcapitata were 5.96 and 2.87 mg L(-1), respectively. The LC50-6d of the ostracod Heterocypris incongruens was 4.84 mg L(-1). A growth inhibition of H. incongruens was registered after carbaryl exposure and the IC20-6d was 1.29 mg L(-1). Our results suggest that the daphnid test sensitivity was better than other used tests. Moreover, carbaryl has harmful and toxic effects on tested species because it acts at low concentrations on diverse life history traits of species and induce embryo deformities in crustaceans.

Investigation of differences in sensitivity between 3 strains of Daphnia magna (crustacean Cladocera) exposed to malathion (organophosphorous pesticide).

Acute and chronic ecotoxic effects of organophosphorous insecticide malathion (Fyfanon 50 EC 500 g L(-1)) were investigated on three strains of Daphnia magna. The nominal effective concentrations immobilizing 50% (EC50) of Daphnia after 24 and 48 h were 0.53 and 0.36 µg L(-1), 0.70 and 0.44 µg L(-1), and 0.75 and 0.46 µg L(-1) for the strains 1, 2, and 3 respectively. There was an increase in malathion ecotoxicity with time of exposure as confirmed by chronic studies. In fact, after 21 days of exposure, significant effects on survival (lowest observed effect concentration or LOECs) were recorded at malathion concentrations of 22, 220 and 230 ng L(-1) for strains 1, 2 and 3 respectively. Other endpoints were also examined, including reproduction (with different parameters), body length and embryo toxicity. ICs10 and ICs20 were calculated for these different parameters. ICs10 ranged from 4.7 to more than 220 ng L(-1) for the three tested strains. The most sensitive parameter was the number of neonates per adult (ICs10 = 4.7 and 10.8 ng L(-1) for strains 1 and 2 respectively) or the number of broods (IC10 = 10 ng L(-1) for strain 3). Moreover, an increase in embryo development abnormalities was observed at the two highest tested malathion concentrations. Abnormalities comprised undeveloped second antennae, curved or unextended shell spines, and curved post-abdomen spines in live neonates. Results suggest that malathion could act as an endocrine disruptor in D. magna as it interferes with development. It also induces a significant decrease in acetylcholinesterase (AChE) activities for the three strains. Both strains 2 and 3 seemed more sensitive (LOECs = 60 ng L(-1)) than strain 1 (LOEC = 120 ng L(-1)). Our results suggest that the AChE activity response can also be used as a biomarker of inter-strain (or inter-clonal) susceptibility (i.e. strain (or clone)-specific response).

Is acetylcholinesterase a biomarker of susceptibility in Daphnia magna (Crustacea, Cladocera) after deltamethrin exposure?

In the present study, we explored the possibility of using the acetylcholinesterase (AChE) as a biomarker after deltamethrin (pyrethroid insecticide) exposure with three strains of the cladoceran Daphnia magna. Four calculated time-weighted deltamethrin concentrations (20.1, 40.3, 80.6 and 161.3 ng L(-1)) were compared against control acetylcholinesterase activity. Our results showed that after 48 h of deltamethrin exposure, all treatments induced a significant decrease of AChE activities whatever the three considered strains. However, diverse responses were registered in terms of lowest observed effect concentrations (LOEC: 80.6 ng L(-1) for strain 1 and 20.1 ng L(-1) for strains 2 and 3) revealing differences in sensitivity among the three tested strains of D. magna. Our results suggest that after deltamethrin exposure, the AChE activity responses can be also used as a biomarker of susceptibility (i.e., variation of strain specific response). Moreover, our results show that strain 1 is the less sensitive in terms of IC50-48 h of AChE, whereas it became the most sensitive when considering the EC50-48 h estimated in the standard ecotoxicity test.

Effect of deltamethrin (pyrethroid insecticide) on two clones of Daphnia magna (Crustacea, Cladocera): a proteomic investigation.

Deltamethrin is a class II pyrethroid insecticide commonly used in agriculture. It is hazardous to freshwater ecosystems, especially for the cladoceran Daphnia magna (Straus 1820). The results of our previous studies based on acute and chronic ecotoxicity experiments revealed differences in the sensitivity between two different clones. In this work, to investigate deltamethrin toxicity mechanisms in two clones of D. magna, we used a proteomic approach in order to analyze changes in protein expression profiles after 48 h of exposure. We detected 1339 spots; then applying statistical criteria (ANOVA p<0.001 and minimum fold change 1.5), only 128 spots were significantly different in the normalized volume. Among the preselected proteins there were 88 up-regulated and 40 down-regulated proteins. Results showed differences in sensitivities after deltamethrin exposure between the clones. Moreover, using the 2-DIGE method, proteomic investigation for deltamethrin exposure proved to be a reliable and powerful approach to investigate effects of deltamethrin as part of research for new metabolic and cellular biomarkers. After identification by mass spectrometry, there were 39 proteins recognized and identified, in which 21 and 18 were up- and down-regulated, respectively, in deltamethrin-exposed clone A compared to three other conditions (controls of each clone and deltamethrin-exposed clone 2). Up- and down-regulated proteins belonged to 12 biological processes (i.e. metabolic processes, apoptosis and stimulus response) and 5 molecular functions (i.e. catalytic activity, binding, structural molecular activity, antioxidant and receptor activities). Identification of these deregulated proteins opens a new way in discovering new molecular targets and putative biomarkers in daphnids exposed to deltamethrin.

Sensitivity of different aquatic bioassays in the assessment of a new natural formicide.

Agrochemicals have the potential to cause deleterious effects on living organisms and therefore they must be subjected to various (eco)toxicological studies and monitoring programs in order to protect human health and the environment. The aim of this study was to assess the ecotoxicity of a new natural formicide with a battery of three classical and three ecotox-kit tests. The former tests were performed with Aliivibrio fischeri bacteria (Lumistox test), the cladoceran Daphnia magna and Pseudokirchneriella subcapitata algae, and the latter with Thamnotoxkit F(TM) (Thamnocephalus platyurus), Ostracodtoxkit F® (Heterocypris incongruens) and LuminoTox (photosynthetic enzyme complexes). In the range of formicide concentrations tested (from 0.06 to 2.0 g L(-1)), the measurement endpoint values varied from 0.79 g L(-1) for the algal test to > 2 g L(-1) for the LuminoTox and Ostracodtoxkit F® tests. Hierarchical sensitivity ranking based on the no-observed effect concentration (NOEC) values established to assess the formicide ecotoxicity was as follows: algal growth inhibition test ≈ daphnid immobilization test ≈ bacterial luminescence inhibition test > Thamnotoxkit F™ > LuminoTox > Ostracodtoxkit F®. Overall, results from the battery of bioassays showed that this formicide preparation presents low ecotoxicity as compared to the aquatic ecotoxicity of presently commercialized formicides. In conclusion, classical aquatic bioassays are more sensitive than ecotox-kit tests in the assessment and monitoring of the new natural formicide.