Using single-species and algal communities to determine long-term adverse effects of silver nanoparticles on freshwater phytoplankton.

The physical and chemical properties of silver nanoparticles (AgNPs) have led to their increasing use in various fields such as medicine, food, and industry. Evidence has proven that AgNPs cause adverse effects in aquatic ecosystems, especially when the release of Ag is prolonged in time. Several studies have shown short-term adverse effects of AgNPs on freshwater phytoplankton, but few studies have analysed the impact of long-term exposures on these populations. Our studies were carried out to assess the effects of AgNPs on growth rate, photosynthesis activity, and reactive oxygen species (ROS) generation on the freshwater green algae Scenedesmus armatus and the cyanobacteria Microcystis aeruginosa, and additionally on microcystin (MC-LR) generation from these cyanobacteria. The tests were conducted both in single-species cultures and in phytoplanktonic communities exposed to 1 ngL-1 AgNPs for 28 days. The results showed that cell growth rate of both single-species cultures decreased significantly at the beginning and progressively reached control-like values at 28 days post-exposure. This effect was similar for the community-cultured cyanobacteria, but not for the green algae, which maintained a sustained decrease in growth rate. While gross photosynthesis (Pg) increased in both strains exposed in single cultures, dark respiration (R) and net photosynthesis (Pn) decreased in S. armatus and M. aeruginosa, respectively. These effects were mitigated when both strains were exposed under community culture conditions. Similarly, the ROS generation shown by both strains exposed in single-species cultures was mitigated when exposure occurred in community cultures. MC-LR production and release were significantly decreased in both single-species and community exposures. These results can supply helpful information to further investigate the potential risks of AgNPs and ultimately help policymakers make better-informed decisions about their utilization for environmental restoration.

Adverse effects of iron-based nanoparticles on freshwater phytoplankton Scenedesmus armatus and Microcystis aeruginosa strains.

Zero-valent nano-iron particles (nZVI) are increasingly present in freshwater aquatic environments due to their numerous applications in environmental remediation. However, despite the broad benefits associated with the use and development of nZVI nanoparticles, the potential risks of introducing them into the aquatic environment need to be considered. Special attention should be focused on primary producer organisms, the basal trophic level, whose impact affects the rest of the food web. Although there are numerous acute studies on the acute effects of these nanoparticles on photosynthetic primary producers, few studies focus on long-term exposures. The present study aimed at assessing the effects of nZVI on growth rate, photosynthesis activity, and reactive oxygen activity (ROS) on the freshwater green algae Scenedesmus armatus and the cyanobacteria Microcystis aeruginosa. Moreover, microcystin production was also evaluated. These parameters were assessed on both organisms singly exposed to 72 h-effective nZVI concentration for 10% maximal response for 28 days. The results showed that the cell growth rate of S. armatus was initially significantly altered and progressively reached control-like values at 28 days post-exposure, while M. aeruginosa did not show any significant difference concerning control values at any time. In both strains dark respiration (R) increased, unlike net photosynthesis (Pn), while gross photosynthesis (Pg) only slightly increased at 7 days of exposure and then became equal to control values at 28 days of exposure. The nZVI nanoparticles generated ROS progressively during the 28 days of exposure in both strains, although their formation was significantly higher on green algae than on cyanobacteria. These data can provide additional information to further investigate the potential risks of nZVI and ultimately help decision-makers make better informed decisions regarding the use of nZVI for environmental remediation.

Heavy metals immobilization capability of two iron-based nanoparticles (nZVI and Fe3O4): Soil and freshwater bioassays to assess ecotoxicological impact.

The contamination by heavy metals constitutes an environmental problem of great importance in the last decades, and demands of society for clean environments are increasingly evident. To achieve this goal, several strategies have appeared for the in situ remediation of soil contamination caused by heavy metals. This study evaluated two types of iron-based nanoparticles, zero-valent iron nanoparticles (nZVI) and Fe3O4 nanoparticles, for the effective immobilization of Furthermore, we conducted a set of ecotoxicological bioassays: Microtox® Test, Caenorhabditis elegans Test, and Phytoplankton Toxicity Tests, on selected soil and aquatic test organisms to both, i) evaluate the potential ecotoxicological risks associated with nanoparticles treatment, and ii) to define sensitive organisms to be used as suitable bioindicators of heavy metals pollution. The application of 5% nZVI significantly reduced the amount of bioavailable heavy metals, which was effective from an ecotoxicity point of view as a reduction of the toxicity of was observed. Among the bioassays used, C. elegans seems the most effective reference organism in detecting changes in the toxicity of and therefore, C. elegans was found to be a sensitive heavy metals pollution bioindicator. When the Combination index (CI) was obtained to determine combined heavy metals interactions, the results indicated that toxicity would be higher than that expected for Pb, Cd and Zn individually considered, due to the proved antagonistic interactions of those toxicants. The obtained results suggested that nZVI nanoparticles are susceptible to be used as a soil remediation strategy for heavy metal pollution, although a short reactive lifespan must be considered, and therefore its effectiveness in long periods remains to be elucidated.

Potential risk of acute toxicity induced by AgI cloud seeding on soil and freshwater biota.

Silver iodide is one of the most common nucleating materials used in cloud seeding. Previous cloud seeding studies have concluded that AgI is not practically bioavailable in the environment but instead remains in soils and sediments such that the free Ag amounts are likely too low to induce a toxicological effect. However, none of these studies has considered the continued use of this practice on the same geographical areas and thus the potential cumulative effect of environmental AgI. The aim of this study is to assess the risk of acute toxicity caused by AgI exposure under laboratory conditions at the concentration expected in the environment after repeated treatments on selected soil and aquatic biota. To achieve the aims, the viability of soil bacteria Bacillus cereus and Pseudomonas stutzeri and the survival of the nematode Caenorhabditis elegans exposed to different silver iodide concentrations have been evaluated. Freshwater green algae Dictyosphaerium chlorelloides and cyanobacteria Microcystis aeruginosa were exposed to silver iodide in culture medium, and their cell viability and photosynthetic activity were evaluated. Additionally, BOD5 exertion and the Microtox® toxicity test were included in the battery of toxicological assays. Both tests exhibited a moderate AgI adverse effect at the highest concentration (12.5µM) tested. However, AgI concentrations below 2.5µM increased BOD5. Although no impact on the growth and survival endpoints in the soil worm C. elegans was recorded after AgI exposures, a moderate decrease in cell viability was found for both of the assessed soil bacterial strains at the studied concentrations. Comparison between the studied species showed that the cyanobacteria were more sensitive than green algae. Exposure to AgI at 0.43µM, the reference value used in monitoring environmental impact, induced a significant decrease in photosynthetic activity that is primarily associated with the respiration (80% inhibition) and, to a lesser extent, the net photosynthesis (40% inhibition) in both strains of phytoplankton and a moderate decrease in soil bacteria viability. These results suggest that AgI from cloud seeding may moderately affect biota living in both terrestrial and aquatic ecosystems if cloud seeding is repeatedly applied in a specific area and large amounts of seeding materials accumulate in the environment.

Interference of heavy metals on the photosynthetic response from a Cr(VI)-resistant Dictyosphaerium chlorelloides strain.

The successful selection of a particular type of bioelement and its association to the appropriate transducer determines the specificity of a biosensor. Therefore, from a strain of chloroficea Dictyosphaerium chlorelloides, modified in laboratory to tolerate high Cr(VI) concentrations, the possible interferences of other heavy metals on photosynthetic activity were studied. After exposing wild type and Cr(VI)-resistant cells to increasing Ag(+1), Co(+2), Hg(+2), Cr(+3), Cu(+2), Zn(+2), Fe(+3) and Cd(+2) concentrations, both photosynthetic quantum yields was compared. Photosynthetic electron transport rates were measured with a TOXY-PAM chlorophyll fluorometer, non-linear regression analysis of each of the toxicity tests was done, and means of both groups were compared using unpaired t test. The results show no significant differences between both cell types when they were exposed to Ag(+1), Co(+2), Hg(+2), Cr(+3), Cu(+2), Fe(+3) and Cd(+2) metal ions, and extremely significant differences (p < 0.0001) to Zn(+2) exposures. These results demonstrate the suitability of this Cr(VI)-resistant type D. chlorelloides strain as a suitable bioelement to be coupled to a biosensor based on dual-head microalgae strategy to detect and quantify Cr(VI) in water courses and waste water treatment plants. However, some disturbance may be expected, especially when certain analyte species such as zinc are present in water samples tested. The analysis of binary mixtures between Zn(+2) and other heavy metals showed a slight antagonistic phenomenon in all cases, which should not alter the potential Zn(+2) interference in the Cr(+6) detection process.

Photosynthetic activity and protein overexpression found in Cr(III)-tolerant cells of the green algae Dictyosphaerium chlorelloides.

Chromium is an important constituent in effluents obtained from chromium plating industries. Due to the highly toxic nature of Cr(VI), attention has been shifted to less hazardous Cr(III) electroplating processes. This study evaluated aquatic toxicity of Cr(III)-containing laboratory samples representative of effluents from chromium electroplating industries, on the photosynthetic activity exhibited by both Cr(III)-sensitive (Dc1M(wt)) and tolerant (Dc1M(Cr(III)R30)) Dictyosphaerium chlorelloides strains. Additionally, selected de novo-determined peptide sequences, obtained from Dc1M(Cr(III)R30), have been analyzed to evidence the possible Cr(III) toxic mechanism involved in the resistance of these cells to high Cr(III) levels in aquatic environments. Dc1M(Cr(III)R30) strain exhibited a gross photosynthetic balance of about five times lower than that exhibited by Dc1M(wt) strain, demonstrating that Dc1M(Cr(III)R30) has a photosynthetic yield significantly lower than Dc1M(wt). SDS-PAGE of Dc1M(Cr(III)R30) samples showed the presence of at least two protein bands (23.05 and 153.46 KDa, respectively) absent in wild-type strain samples. Although it has achieved a low coincidence between the lower molecular weight band and a GTPase identified from genome of the green alga Chlamydomonas reinhardtii, none of de novo peptide sequences obtained showed a significant MS-BLAST score, so that further studies will be required.

Bioadsorption and bioaccumulation of chromium trivalent in Cr(III)-tolerant microalgae: a mechanisms for chromium resistance.

Anthropogenic activity constantly releases heavy metals into the environment. The heavy metal chromium has a wide industrial use and exists in two stable oxidation states: trivalent and hexavalent. While hexavalent chromium uptake in plant cells has been reported that an active process by carrying essential anions, the cation Cr(III) appears to be taken up inactively. Dictyosphaerium chlorelloides (Dc1M), an unicellular green alga is a well-studied cell biological model organism. The present study was carried out to investigate the toxic effect of chromium exposures on wild-type Cr(III)-sensitive (Dc1M(wt)) and Cr(III)-tolerant (Dc1M(Cr(III)R30)) strains of these green algae, and to determine the potential mechanism of chromium resistance. Using cell growth as endpoint to determine Cr(III)-sensitivity, the IC50(72) values obtained show significant differences of sensitivity between wild type and Cr(III)-tolerant cells. Scanning electron microscopy (SEM) showed significant morphological differences between both strains, such as decrease in cell size or reducing the coefficient of form; and transmission electron microscopy (TEM) revealed ultrastructural changes such as increased vacuolization and cell wall thickening in the Cr(III)-tolerant strain with respect to the wild-type strain. Energy dispersive X-ray analysis (SEM/XEDS) revealed that Cr(III)-tolerant D. chlorelloides cells are able to accumulate considerable amounts of chromium distributed in cell wall (bioadsorption) as well as in cytoplasm, vacuoles, and chloroplast (bio-accumulation). Morphological changes of Cr(III)-tolerant D. chlorelloides cells and the presence of these electron-dense bodies in their cell structures can be understood as a Cr(III) detoxification mechanism.

Toxic risk associated with sporadic occurrences of Microcystis aeruginosa blooms from tidal rivers in marine and estuarine ecosystems and its impact on Artemia franciscana nauplii populations.

Microcystis aeruginosa is a species of freshwater cyanobacteria which can form harmful algal blooms in freshwater water bodies worldwide. However, in spite its sporadic occurrences for short periods of time in estuarine waters, their influence on zooplankton populations present in these ecosystems has not been extensively studied. In this work, Artemia franciscana was used as test organism model, studying mortality against several strains of M. aeruginosa with different degrees of toxigenicity, measuring whole-live cells and homogenate extracts. Results were compared with microcystin-LR equivalent content, measured by immunoassay. The results show that there were no significant differences between both exposure models (whole cells and extracts), and there are significant differences respect to the toxigenicity of cyanobacterial blooms depending of the M. aerugionosa strain involved in the process. Analysis of microcystin-LR equivalent concentration test immediately below the lowest significant concentration in all M. aerugionosa strains was used to determine the potential risk associated with the cell densities during a bloom. Comparison among the selected M. aerugionsa strains show that these factors have influence in the results obtained, and thus, several differences have been evidenced depending of the microcystin-LR equivalent production and the strain type involved.

Cytotoxicity and genotoxicity of sewage treatment plant effluents in rainbow trout cells (RTG-2).

The cytotoxicity and genotoxicity of 11 organic fractions from sewage treatment plant (STP) effluents were tested using the RTG-2 rainbow trout permanent cell line. An automated in vitro micronucleus assay developed for RTG-2 cells was used to test the genotoxicity, whereas neutral red uptake, kenacid blue protein assay and ATP content were used to evaluate cytotoxicity. The induction of micronuclei (MN) and alterations in the cell cycle were analysed in these cells by flow cytometry after exposure to the organic fractions for 72 h. More than half of the organic extracts tested demonstrated a significant increase in the MN frequency, thus indicating that most of them can be considered to be genotoxic. The extracts were analysed chemically by GC/MS. Although the most frequently detected compounds in the effluents were bisphenol A (BPA), octylphenol (OP), di-2-ethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP), as well as other possible mutagens, the concentrations cannot explain the genotoxicity of the individual chemicals, thereby suggesting a mixture effect. The results obtained support the need to apply effect-based tests to monitor complex mixtures as the most accurate means of assessing the genotoxicity of environmental samples.

Importance of strain type to predict the toxicological risk associated with Microcystis aeruginosa blooms: comparison of Microtox(®) analysis and immunoassay.

The occurrence of toxic cyanobacterial blooms in aquatic environments, associated with human health problems and animal deaths, has increased the need for rapid, reliable and sensitive methods to determine the toxicity of microcystin produced by cyanobacteria. An in vitro Microtox(®) system and a commercially available microcystin ELISA were used to screen out the potential risk associated with selected Microcystis aeruginosa strains (Ma1D-Ma8D). Results showed the existence of three differentiated groups in the selected M. aeruginosa strains. Strains Ma7D and Ma6D were determined to be very toxic, strains Ma2D, Ma1D and Ma5D as moderately toxic and strains Ma8D, Ma4D and MA3D as non-toxic. These results agreed with the microcystin concentration values obtained by immunoassay. Although the data obtained by other authors clearly show that Microtox(®) is not sensitive to microcystins, our results suggested that this bioluminescence assay may prove useful in the preliminary screening of cyanobacterial blooms for microcystin-based toxicity. Additionally, the combination of immunodetection and toxicity-based Microtox(®) provides a useful addition to the methods already available for detection of cyanobacterial toxins.

Toxic effects and specific chromium acquired resistance in selected strains of Dyctiosphaerium chlorelloides.

Due to its various uses, chromium contamination has become widespread in a diverse array of environments. The present study was carried out to investigate the toxic effect of chromium exposures on sensitive and resistant strains of the green algae Dyctiosphaerium chlorelloides, and to determine the nature and mechanism of chromium-resistant cells that arise. The toxic effect on the photosynthetic performance of chromium exposures in both cell populations, and the sensitive differences due to chromium oxidation state, were estimated, and the results indicate that although the photosynthetic performance in both strains were inhibited, there are not significant differences among IC(50(72)) values obtained in toxicity assays with both chromium oxidation states in wild-type cells, and however these differences are very significant when the assays were performed with Cr(VI) resistant cells. The 72-h 50% inhibitory concentration values obtained with Cr(III) exposures were similar for both strains. Additionally, by means of the SEM/EDX and TEM microscopic techniques, the occurrence of rapid morphological evolution in the microalgal cells and the possible detoxificant mechanisms was observed after exposure of the wild strain to chromium hexavalent. Moreover, the different response in photosynthetic activity observed between sensitive and resistant cells of D. chlorelloides in the presence of Cr(VI) and Cr(III) could be used to obtain a chromium-specific eukaryotic microalgal biosensor.

Toxic effect and adaptation in Scenedesmus intermedius to anthropogenic chloramphenicol contamination: genetic versus physiological mechanisms to rapid acquisition of xenobiotic resistance.

Anthropogenic water pollution is producing a challenge to the survival of phytoplankton populations. From an ecological point of view, the tolerance of these microorganisms to water pollution is of paramount importance since they are the principal primary producers of aquatic ecosystems. The adaptation of a common chlorophyta species (Scenedesmus intermedius) exposed to selected dose-response chloramphenicol (CAP) concentrations has been analyzed. A fluctuation analysis demonstrated that CAP-resistant cells arise due to spontaneous mutation which occurs randomly prior to the antibiotic exposure. CAP-inhibited growth and photosynthetic performance of algal cells at 0.28 mg/l, and the IC(50(72)) value was established in 0.10 mg/l for both parameters. The mutation rate from CAP sensitivity to resistance was 1.01 x 10(-5) mutations per cell division, while the frequency of CAP-resistant allele in non-polluted environment was estimated to be 5.5 CAP-resistant mutants per 10(3) sensitive-cells. These results demonstrate that resistant mutants exhibit a diminished fitness until 5 mg/l of CAP, thus enabling the survival of microalgae population.

Toxicity and characterization of cholinesterase-inhibition induced by diisopropyl fluorophosphate in Artemia salina larvae.

The acute toxicity of diisopropyl fluorophosphate (DFP) on three age classes of Artemia salina was evaluated. An increase in toxicity of this organophosphorous (OP) compound was found following longer development of A. salina larvae. The effects of pretreatment with the non-selective muscarinic antagonist atropine, the two reversible acetylcholinesterase inhibitors physostigmine and pyridostigmine, and the cholinesterase-reactivating oxime 2-pyridine aldoxime methoiodide (2-PAM), as individual and combined pretreatments, on DFP-induced lethality in 24h Artemia were also investigated. The lethal action of DFP was not prevented by pretreatment of 24h Artemia with atropine, physostigmine, and pyridostigmine, while 2-PAM proved effective against intoxication with this OP compound. The inhibitory effects of combinations of atropine (10(-5)M) plus 2-PAM or physostigmine were greater than those elicited by either drug alone, with the maximum protection afforded being 100%. Pretreatment with 2-PAM (10(-6)M) plus physostigmine or pyridostigmine was ineffective. These results suggest that the combinations of atropine plus 2-PAM or physostigmine are effective in the prevention of the lethal effects induced by DFP in A. salina larvae.

Toxic effects induced by salt stress on selected freshwater prokaryotic and eukaryotic microalgal species.

In order to determine the short-term impact induced by salt stress, cultures of Dictyosphaerium chlorelloides and Microcystis aeruginosa were grown in presence of increasing sea-salt concentrations. Growth rate and photosystem II activity in D. chlorelloides, and photosynthetic oxygen production (in both species) were analyzed. A concentration-dependent response was obtained with the presence of sea-salt in culture medium, being M. aeruginosa (EC(50(72)) = 76.6 mM) more sensitive to salt stress than D. chlorelloides (EC(50(72)) = 340.7 mM). However, comparative analysis between growth and Phi(PSII) inhibition in D. chlorelloides shown that there are not significant differences among EC(50(72)) values obtained. An immediate toxic response, induced by increase of sea-salt concentration, has been obtained applying the calculated EC(50(72)) values in both species. These results shown that sea-salt acts as a sensitive and rapid toxic compound in algal cells, and that the sensitivity of M. aeruginosa to salinity stress is much higher than that of D. chlorelloides.

Cytotoxic and genotoxic effect in RTG-2 cell line exposed to selected biocides used in the disinfection of cooling towers.

The cytotoxic and genotoxic effects induced by trichloroisocyanuric acid, Oxone, and sodium bromide, active principles included in formulations for cleaning and disinfection of cooling towers, were studied on RTG-2 cell line. Neutral red assay was used to determine the cellular viability. Toxicity ranking based on IC(50) values found that trichloroisocyanuric acid was the most cytotoxic biocide tested followed by Oxone, whereas sodium bromide resulted in a very low cytotoxicity. DNA damage has been evaluated on RTG-2 cultures by means of an in vitro assay based on the ability of PicoGreen fluorochrome to interact preferentially with dsDNA, and the results indicated that trichloroisocyanuric acid induced DNA strand breaks at concentrations above 1.2 mg/l, equivalent to 1/50-EC(50(48)), whereas exposures to Oxone and sodium bromide did not induce DNA damage at the maximal concentrations tested (1/10-EC(50(48))). These results confirm the suitability of this method for the screening of genotoxic effects of this type of aquatic pollutants, and we suggest their use in environmental risk assessment procedures.

Inhibition of growth and photosynthesis of selected green microalgae as tools to evaluate toxicity of dodecylethyldimethyl-ammonium bromide.

The effect of dodecylethyldimethyl-ammonium bromide (DEAB), a quaternary ammonium, compound widely used as disinfectant, on phytoplankton of inland water systems was analysed by using an experimental model. A toxicity test was based on inhibition of photosynthesis performances (effective quantum yield from photosystem II, Phi(PSII) and O(2) production) of the phytoplanktonic species Scenedesmus intermedius and Dictiosphaerium chlorelloides (Chlorophyceae) under growing doses of DEAB. A concentration-dependent toxic response was obtained in both parameters analysed. In addition, this response was almost immediate. Consequently, the measurement of both parameters (Phi(PSII )and O(2) production) allows to assess DEAB toxicity with higher standards of precision and repeatability. We propose that this procedure could be used to detect presence of quaternary ammonium pollutants in freshwater.

Genotoxic effects of selected biocides on RTG-2 fish cells by means of a modified Fast Micromethod Assay.

A sensitive in vitro assay for detecting DNA damage in RTG-2 cells culture is described. This assay employs a dye, PicoGreen double stranded DNA (dsDNA) quantitation reagent, which becomes intensely fluorescent upon binding nucleic acids. The assay includes a simple and rapid 50-min sample lysis in the presence of EDTA, SDS, and high urea concentration at pH 10, followed by time-dependent DNA denaturation at pH 11.6 after NaOH addition. The time course and the extent of DNA denaturation are followed in a microplate fluorescence reader at room temperature for less than 1h. Comparative studies between suspension and fixed RTG-2 cells indicated that it is possible to apply this methodology in both cases with good results. Neutral red assay was used for to determine the cellular viability when RTG-2 cultures were exposed to tetrakis(hydroxymethyl) phosphonium chloride (THPC) and benzalkonium chloride (BC), as biocides used in the disinfection of cooling towers. The results obtained by neutral red assay indicate IC(50(48)) values of 0.017 (0.011-0.028) and 2.71 (1.91-3.86) mg/L for tetrakis(hydroxymethyl) phosphonium chloride and benzalkonium chloride, respectively. DNA damage has been evaluated for both disinfectants in RTG-2 culture, by exposure to 1/10-, 1/25-, 1/50-, and 1/100-IC(50(48)) value, and the results obtained indicate a strain scission factor (SSF) of 0.126+/-0.014, 0.181+/-0.014, 0.217+/-0.013, and 0.245+/-0.013 in cell suspensions, and 0.077+/-0.019, 0.107+/-0.014, 0.151+/-0.014, and 0.202+/-0.015 in attached cells for tetrakis(hydroxymethyl) phosphonium chloride; while the SSF values for benzalkonium chloride are 0.023+/-0.009, 0.033+/-0.017, 0.068+/-0.012, and 0.088+/-0.015 in cell suspensions, and 0.033+/-0.010, 0.044+/-0.011, 0.080+/-0.009, and 0.093+/-0.010 in attached cells. Thus, the assay proposed in this study has made it possible to show DNA damage in RTG-2 cells when exposed to 0.2(1/100 IC(50(48))) and 300(1/10 IC(50(48))) Hg/L of tetrakis(hydroxymethyl) phosphonium chloride and benzalkonium chloride, respectively. The results obtained indicate that the Fast Micromethod Assay, applied on RTG-2 cell line cultures, is a fast and sensitive method for the early DNA damage detection in the aquatic environment.

Comparative study on the environmental risk induced by several pyrethroids in estuarine and freshwater invertebrate organisms.

The acute toxicity of permethrin, resmethrin, and cypermethrin to four species of aquatic non-target invertebrate organisms, found in estuarine and freshwater ecosystems, was evaluated. Artemia franciscana and Brachionus plicatilis larvae, as estuarine organisms, and Brachionus calyciflorus and Thamnocephalus platyurus larvae, as freshwater organisms, were exposed for 24 h to concentrations of these pyrethroids, and the LC(50) values were compared. The freshwater organisms were more sensitive to these pyrethroids than estuarine organisms tested. A. franciscana larvae were more tolerant organisms than B. plicatilis larvae. The freshwater organisms tested have demonstrated to be a good alternative to the standard acute toxicity assays using Daphnia, although Brachionus plycatilis larvae were more sensitive to these pyrethroid insecticides than T. platyurus. Analysis of 24 h LC(50) values of these pyrethroids, determined by static bioassays, revealed that the rank order of toxicity was: permethrin

The use of carbamates, atropine, and 2-pyridine aldoxime methoiodide in the protection of Artemia salina against poisoning by carbophenothion.

The acute toxicity of carbophenothion to three age classes of Artemia salina was evaluated. An increase in toxicity of carbophenothion was found following longer development of A. salina. The effect of pretreatment with the nonselective muscarinic antagonist atropine, the two reversible acetylcholinesterase-inhibitors physostigmine and pyridostigmine, and the cholinesterase-reactivating oxime 2-pyridine aldoxime methochloride (2-PAM) on carbophenothion-induced lethality in 24-h-old A. salina was also investigated. The lethal action of carbophenothion was completely prevented by pretreatment of A. salina with 2-PAM. Atropine and pyridostigmine afforded a maximal protection of approximately 87% and 72%, respectively, compared to control values. In contrast, physostigmine was ineffective. The inhibitory effects of combinations of 10(-5) M atropine with physostigmine, pyridostigmine, or 2-PAM were greater than those elicited by either drug alone, with the maximum protection afforded being 92.58%, 100%, and 100%, respectively. In the presence of 10(-7) M atropine, neither pyridostigmine nor 2-PAM provided additional inhibition of the lethality compared to that with either drug alone, whereas the protection afforded by 10(-7) M atropine plus physostigmine increased as the concentration of carbamate increased (up to 10(-3) M). Pretreatment with pyridostigmine or physostigmine plus 2-PAM (10(-6) M) slightly enhanced the maximal inhibition of carbophenothion lethality compared to that with either drug alone. It is suggested that the most active combined pretreatment studied here was physostigmine plus atropine.