Acute toxicity of three detergents and two insecticides in the lugworm, Arenicola marina (L.): a histological and a scanning electron microscopic study

The toxicity of three detergents (sodium dodecyl benzene sulfonate, sodium dodecyl sulfate and Triton X-100) and two insecticides (Carbaryl and Parathion-ethyl) on the lugworm, Arenicola marina, was investigated. The 48-h LC₅₀ values were established and the morphological alterations in epidermis, gills and intestine were analysed by light and scanning electron microscopy. The three detergents were equally toxic (LC₅₀ from 12 to 15 mg · l⁻¹) while the insecticides were more potent (LC₅₀ = 7.2 and 2.7 mg · l⁻¹, for the Carbaryl and the Parathion-ethyl, respectively). The gills and the epidermic receptors were the most sensitive sites of the lugworm, while the thoracic epidermis was the most resistant of the structures studied.     

Protection against the toxicity of microcystin-LR and cylindrospermopsin in Artemia salina and Daphnia spp. by pre-treatment with cyanobacterial lipopolysaccharide (LPS)

Purified cyanobacterial lipopolysaccharide (LPS) was not acutely toxic to three aquatic invertebrates (Artemia salina, Daphnia magna and Daphnia galeata) in immersion trials. However, pre-exposure (24 h) to 2 ng mL⁻¹ LPS increased the LC₅₀ of microcystin-LR significantly in all 3 species. Similar results were observed with A. salina pre-treated with the same concentration of cyanobacterial LPS and subsequently exposed to cylindrospermopsin, increasing the LC₅₀ by 8. The findings indicate the need to include exposures to defined combinations of cyanotoxins, and in defined sequences, to understand the contributions of individual cyanotoxins in accounting for cyanobacterial toxicity to invertebrates in natural aquatic environments.     

Larvicidal action of ethanolic extracts from fruit endocarps of Melia azedarach and Azadirachta indica against the dengue mosquito Aedes aegypti

Ethanolic extracts from the kernels of ripe fruits from the Indian Lilac Melia azedarach and from the well-known Neem tree, Azadirachta indica were assayed against larvae of Aedes aegypti, the mosquito vector of dengue fever. The lethality bioassays were carried out according to the recommendations of the World Health Organization. Extracts were tested at doses ranging from 0.0033 to 0.05 g% in an aqueous medium for 24 and 48 h, at 25 or 30 °C, with or without feeding of the larvae. LC₅₀, LC₉₅ and LC₉₉ were determined. Both seed extracts proved lethal for third to fourth instar larvae. Non-fed A. aegypti larvae were more susceptible to Azadirachta extracts at both temperatures. Under a more realistic environmental situation, namely with fed larvae at 25 °C, the death rates caused by the Melia extract were higher, although at 30 °C the extract of Azadirachta had an even higher lethality. Inter allia, the LC₅₀ values for the crude extracts of these two members of the Meliaceae ranged from 0.017 to 0.034 g% while the LC₉₉ values ranged from 0.133 to 0.189 g%. Since no downstream processing was undertaken to purify the active agents in the extracts, our findings seem very promising, suggesting that it may be possible to increase the larvicidal activity further by improving the extraction and the fractionation of the crude limonoids, for instance removing the co-extracted natural fats.     

Evaluation of acute toxicity of carbaryl and malathion to freshwater teleosts, Channa punctatus (Bloch) and Heteropneustes fossilis (Bloch)

Bioassay tests evaluated the acute toxicity of carbaryl (a carbamate) (50'% wettable powder, W.P.), and malathion (50% E.C.) (an organophosphate) (50% emulsifiable concentrate; E.C.) by determining their LC₅₀ and the acute toxic ranges for 24, 48 and 72, and 96-h exposure to Channa punctatus (Bl.) and Heteropneustes fossilis (Bl.). Regression equations and slope functions were determined for different time periods. The relative susceptibility of the fish, the relative toxicity, and the safe concentrations of these biocides were calculated on the basis of LC₅₀ for 96 h. C. punctatus was found to be relatively more susceptible to both the compounds, while malathion was the more toxic of the two biocides.     

Toxicity assessment of fumonisins using the brine shrimp (Artemia salina) bioassay.

The Fusarium mycotoxins fumonisin B₁ (FB₁) (1) and B₂ (FB₂) (2), their hydrolysed analogues HFB₁ (3) and HFB₂ (4) and the recently discovered fumonisin derivatives N-palmitoyl-HFB₁ (5) and N-carboxymethyl-FB₁ (6) were compared for their toxicity in a short term bioassay using brine shrimp (Artemia salina). The brine shrimp were hatched in artificial sea water and exposed to the fumonisins in microwell plates with a mortality endpoint after 48 hours. LC₅₀ values were calculated after Probit transformation of the resulting data. Of the substances tested, fumonisin B₁ emerged to be the most toxic whereas its N-carboxymethyl analogue was 100-fold less effective. The hydrolysed fumonisins showed a four- to sixfold reduced toxicity compared to FB₁. N-Palmitoyl-HFB₁ had a higher LC₅₀ value than its precursor HFB₁. The brine shrimp assay proved to be a convenient and rapid system for toxicity assessment of this group of mycotoxins.     

Inhibition by selenium compounds of catecholamine secretion due to inhibition of Ca2+ influx in cultured bovine adrenal chromaffin cells

Selenium is an essential trace metal element, whereas large doses of selenium exert adverse effects to the human body. We examined the effects of selenium compounds, sodium selenite (Na2SeO3) and sodium selenate (Na2SeO4), on catecholamine secretion from cultured bovine adrenal chromaffin cells. Treatment of chromaffin cells with sodium selenite for 72, 48, and 24 h caused decreases in protein and catecholamine contents, in association with cell damage, at concentrations over 30, 300, and 300 microM, respectively. The cells treated with subtoxic conditions (<100 microM, 48 h) of sodium selenite were used for further experiments. Sodium selenite treatment for 48 h inhibited carbachol (CCh)-induced catecholamine secretion in a concentration-dependent and non-competitive manner, while it did not affect high K+- and veratridine-induced catecholamine secretion. Sodium selenite (100 microM) did not affect CCh- and veratridine-induced 22Na+ influx, while the compound inhibited 45Ca2+ influx induced only by CCh, but not high K+ and veratridine. Sodium selenate even at higher concentrations (1000 microM) did not affect any stimulus-induced catecholamine secretion and 45Ca2+ influx. Thus, sodium selenite may specifically exert adverse effects, such as inhibition of physiological stimulus-induced catecholamine secretion from adrenal chromaffin cells due to inhibition of Ca2+ influx.     

Effect of selenate on growth and photosynthesis of Chlamydomonas reinhardtii

Algal communities play a crucial role in aquatic food webs by facilitating the transfer of dissolved inorganic selenium (both an essential trace element and a toxic compound for a wide variety of organisms) to higher trophic levels. The dominant inorganic chemical species of selenium in freshwaters are selenite (SeO(3)(2-)) and selenate (SeO(4)(2-)). At environmental concentrations, selenite is not likely to have direct toxic effects on phytoplankton growth [Morlon, H., Fortin, C., Floriani, M., Adam, C., Garnier-Laplace, J., Boudou, A., 2005a. Toxicity of selenite in the unicellular green alga Chlamydomonas reinharditii: comparison between effects at the population and sub-cellular level. Aquat. Toxicol. 73(1), 65-78]. The effects of selenate, on the other hand, are poorly documented. We studied the effects of selenate on Chlamydomonas reinhardtii growth (a common parameter in phytotoxicity tests). Growth inhibition (96-h IC(50)) was observed at 4.5+/-0.2 microM selenate (p<0.001), an effective concentration which is low compared to environmental concentrations. Growth inhibition at high selenium concentrations may result from impaired photosynthesis. This is why we also studied the effects of selenate on the photosynthetic process (not previously assessed in this species to our knowledge) as well as selenate's effects on cell ultrastructure. The observed ultrastructural damage (chloroplast alterations, loss of appressed domains) confirmed that chloroplasts are important targets in the mechanism of selenium toxicity. Furthermore, the inhibition of photosynthetic electron transport evaluated by chlorophyll fluorescence induction confirmed this hypothesis and demonstrated that selenate disrupts the photosynthetic electron chain. Compared to the classical 'growth inhibition' parameter used in phytotoxicity tests, cell diameter and operational photosynthetic yield were more sensitive and may be convenient tools for selenate toxicity assessment in non-target plants.     

Lethal and sublethal effects of malathion on three life stages of the grass shrimp, Palaemonetes pugio

The effects of malathion exposure on three life stages of the grass shrimp (Palaemonetes pugio) were evaluated. After 96-h exposures, malathion was most toxic to newly hatched larvae with an LC₅₀ of 9.06 μg l⁻¹ followed by an LC₅₀ of 13.24 μg l⁻¹ for 18-day-old larvae and an LC₅₀ of 38.19 μg l⁻¹ for adult shrimp. In a separate bioassay, to simulate field conditions, newly hatched larvae were exposed to malathion at 6 h day⁻¹ every 5 days at a salinity of 10‰ until metamorphosis to postlarvae. After four pulse dose exposures, mortality was highest in the two highest concentrations, of 15.0 and 30.0 μg l⁻¹. The number of instars to postlarvae was significantly lower in the highest concentration compared to control. The findings indicate that malathion may not directly affect growth in a measurable way but may alter natural metamorphic rhythms at the highest concentrations. Whole body acetylcholinesterase (AChE) activity was measured on Day 0 and Day 15 from the pulse exposure test. AChE activities were not significantly different from controls. Other factors than just AChE inhibition may have contributed to malathion toxicity.     

Toxicity of nutritionally available selenium compounds in primary and transformed hepatocytes

The essential trace element selenium is also toxic at low doses. Since supplementation of selenium is discussed as cancer prophylaxis, we investigated whether or not bioavailable selenium compounds are selectively toxic on malignant cells by comparing primary and transformed liver cells as to the extent and mode of cell death. Sodium selenite and selenate exclusively induced necrosis in a concentration-dependent manner in all cell types investigated. In primary murine hepatocytes, the EC50 was 20 microM for selenite, 270 microM for selenate, and 30 microM for Se-methionine. In the human carcinoma cell line HepG2, the EC50 for selenite was 40 microM, and for selenate 1.1 mM, whereas Se-methionine was essentially non-toxic up to 10 mM. Similar results were found in murine Hepa1-6 cells. Exposure of primary murine cells to selenate or selenite resulted in increased lipid peroxidation. Toxicity was inhibited by superoxide dismutase plus catalase, indicating an important role for reactive oxygen intermediates. In primary hepatocytes, metabolical depletion of intracellular ATP by the ketohexose tagatose, significantly decreased the cytotoxicity of Se-methionine, while the one of selenite was increased. These data do not provide any in vitro evidence that bioavailable selenium compounds induce preferentially apoptotic cell death or selectively kill transformed hepatocytes.     

In vitro cyto- and genotoxicity of organomercurials to cells in culture

The sequence of cytotoxic effects for a series of mercury compounds to the BG/F epithelioid cells derived from fin tissue of bluegill sunfish was phenyl mercuric chloride > methyl mercuric chloride > ethyl mercuric chloride mercuric chloride. This sequence of in vitro cytotoxicity was similar to that observed in a 48-h LC₅₀ in vivo acute toxicity assay with rainbow trout. Using induction of micronuclei as an indicator of genetic damage, the organomercurials, but not mercuric chloride, were noted to be clastogenic to the BG/F cells.     

High grazer toxicity of [D-Asp(3),(E)-Dhb(7)]microcystin-RR of Planktothrix rubescens as compared to different microcystins.

Planktothrix rubescens, the dominant cyanobacterium in Lake Zürich, is generally considered to be toxic to zooplankton. The major toxin was determined by NMR spectroscopy and chemical analysis to be [D-Asp³,(E)-Dhb⁷]microcystin-RR. The compound was isolated in high purity, and its 24-h acute grazer toxicity was compared with microcystin-LR, microcystin-RR, microcystin-YR, and nodularin using a Thamnocephalus platyurus bioassay. Based on LC₅₀ values [D-Asp³,(E)-Dhb⁷]microcystin-RR was the most toxic microcystin tested. Nodularin was slightly more toxic under the conditions of the assay. The large number of individuals available for the grazer bioassay allowed the determination of dose-response curves of the different microcystins. These curves showed marked differences in their steepness. Microcystin-RR, which had nearly the same LC₅₀ as microcystin-LR and microcystin-YR, exhibited a very flat dose-response curve. This flat curve indicates that, for some individuals, lower concentrations of this microcystin are much more toxic than are the other two microcystins. Mortality of 100% requires much higher concentrations of microcystin-RR, indicating the resistance of some animals to the toxin. The purified [D-Asp³,(E)-Dhb⁷]microcystin-RR exhibited a higher molar absorption coefficient determined by quantitative amino acid analysis than the coefficients generally used for other microcystins. This observation has consequences for the risk assessment for microcystins and makes a structural determination of microcystins an absolute requirement. The presence of the dehydrobutyrine residue may be the reason for the higher specific toxicity of [D-Asp³,(E)-Dhb⁷]microcystin-RR when compared to the N-methyldehydroalanine-containing microcystins.     

Behavioural and gill histopathological effects of acute exposure to sodium chloride in moneda (Metynnis orinocensis)

To evaluate the toxicity of sodium chloride (NaCl), juveniles and adult Metynnis orinocensis were exposed for 96 h to 0, 5, 10, 15, 20 or 40 g L⁻¹ of salt. Food intake, behaviour, opercular frequency (OF), mortality, body weight and gill microscopic alterations were evaluated. Behavioural changes were observed in fish exposed to concentrations higher than 10 g L⁻¹. Juveniles and adults showed a progressive decrease in the OF and body weight. Food intake decreased in concentrations below 15 g L⁻¹. Juveniles and adults exposed to 15, 20 or 40 g L⁻¹ had 100% mortality. Lamellar congestion, hyperplasia and fusion were the common microscopic alterations at higher concentrations. The gill congestion severity increased with salt concentration. The LC₅₀ for juveniles and adults were 10.5 g L⁻¹ and 10.8 g L⁻¹, respectively. These results suggest that salt concentrations lower than 5 g L⁻¹ are safe for preventive and therapeutic practices in Metynnis orinocensis; whereas prolonged exposure higher than 10 g L⁻¹ is deleterious in this species.     

Effects of ascorbic acid on selenium teratogenicity in cultured rat embryos

The effects of ascorbic acid, as an exogenous antioxidant, on selenium (Se) teratogenicity were examined using rat embryo culture. Rat embryos at day 9.5 of gestation were cultured for 48 h in the presence of sodium selenite at 10 and 20 microM or sodium selenate at 30 and 100 microM with or without the addition of 1 mM of L-ascorbic acid (AsA). Selenite or selenate alone increased the incidence of embryonic malformation. With AsA, the incidence of selenite-induced embryonic malformation was increased. On the contrary, the incidence of selenate-induced embryonic malformation was decreased with AsA. It was considered from these results that the redox states in the embryonic environment and of Se are critical in Se teratogenicity.     

Studies on selenium-related compounds VI. Biosynthesis of dimethyl selenide in rat liver after oral administration of sodium selenate

Experiments were made to obtain data on the biological action of selenium in order to establish a standard for water quality for public water supply. Biosynthesis of dimethyl selenide in rat liver after oral administration of Na₂SeO₄ was investigated and the volatile selenium formed was identified. The study showed that dimethyl selenide, as a respiratory metabolite, was probably formed in the rat liver. Differences were noted as to dimethyl selenide formation from sodium selenite and sodium selenate in vitro. The test of single oral administration of sodium selenate indicated that dimethyl selenide formation increased progressively up to about 6 mg/kg and then reached a plateau at this dose. The increased accumulation of selenium in the liver after continuous oral administration was found to stimulate the methylation of selenium to dimethyl selenide. When sodium selenate was orally administered to rats, (CH₃)₂Se was found by TLC, GLC, and GC-mass spectrometry.     

In vitro enhancement of erythrocyte merucry uptake by spironolactone.

Four groups of male Wistar rats were fed the following regimen for 40 days: (1) 20 ppm methylmercury chloride (MMC); (2) 20 ppm MMC + 3 ppm sodium selenite; (3) 3 ppm sodium selenite; (4) basal diet. The basal diet which contained 0.4 ppm “organic selenium” originating mainly from fish meal and wheat was resumed on day 41. Protective effect of selenite over toxicity of methylmercury was observed in terms of both growth rate and morbidity.Concentrations of total mercury, methylmercury and selenium were determined on Days 0, 20, 41, 47, 54, and 61 in the brain, liver, kidney, and blood. It was noted that methylmercury increased accumulation of selenium in all the organs analyzed while selenium retention varied according to the type of selenium and the organs. Modification by selenite, despite its protective effect, remained equivocal in regard to the organ accumulation of mercury and its retention therein.     

Chromosome damage induced by selenium salts in human peripheral lymphocytes.

Two inorganic salts of selenium, sodium selenite (Na(2)SeO(3)) and sodium selenate (Na(2)SeO(4)), were screened for damage to chromosome and cell division following exposure to human lymphocyte cultures. In vitro exposure of human peripheral blood lymphocytes to high concentration of two inorganic salts of selenium-sodium selenite (2.9 x 10(-5) M) and sodium selenate (2.65 x 10(-5) M)-was found to be lethal; no blast cells being formed. Lower concentrations of both salts, 5.8 x 10(-6) M and 1.06 x 10(-5) M, respectively, were highly mitostatic. Lower concentrations of sodium selenite (2.9 x 10(-6) M, 1.16 x 10(-6) M and 2.32 x 10(-7) M) and sodium selenate (5.3 x 10(-6) M, 2.65 x 10(-6) M and 1.06 x 10(-6) M), respectively, induced chromosomal aberrations and reduced cell division in proportions directly related to the dose. Sodium selenite was considerably more clastogenic than sodium selenate.     

Effect of selenium on the uptake of methyl mercury across perfused gills of rainbow trout Oncorhynchus mykiss

The uptake of methyl mercury was measured across the perfused gills of rainbow trout Oncorhynchus mykiss. The effect of selenium, either in the blood (perfusion medium), or in the water was investigated. Methyl mercury was effectively taken up from the water across the gills into the perfusate. The uptake rate reached a stable level after 30 min perfusion. When the gills were placed in mercury free water after exposure to mercury in the water for 1 h, they continued to liberate significant amounts of accumulated mercury into the perfusate. Exposure to selenite (SeIV) or selenate (VI) (0.075–0.75 μM) in the external medium did not affect the uptake of methyl mercury across the gills or the liberation of the metal from the gills. Internal selenite or selenate (7.5 μM) augmented the uptake of methyl mercury across the gills and internal selenite also increased the amounts of liberated methyl mercury from the gills in the unload period. Internal selenium, increased the mercury accumulation in the gills, whereas, external selenium did not alter the mercury accumulation in the gills. Uptake of selenium from the water across the gills occurred very slowly.     

The toxicity of the mutagen ‘MX’ and its analogue, mucochloric acid, to rainbow trout hepatocytes and gill epithelial cells and to Daphnia magna

The cytotoxicity of the, in Salmonella, potent mutagenic compound, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) and its structural analogue 3,4-dichloro-5-hydroxy-2[5H]-furanone (mucochloric acid, MCA), was studied in freshly isolated rainbow trout hepatocytes and gill epithelial cells by determining ⁸⁶Rb-leakage and decrease in fluorescence intensity in calcein AM-loaded cells. The acute toxicity of the compounds to Daphnia magna was studied by determining the concentration causing immobilization of the organism. MX proved to be more toxic than MCA both in the cellular assays and in the acute toxicity test with D. magna. MX was more toxic to hepatocytes than to gill epithelial cells. The uptake of [¹⁴C]MX was also much more efficient in hepatocytes than in gill epithelial cells. The uptake of [¹⁴C]MX in hepatocytes was not inhibited by taurocholic acid in excess, indicating that MX is not taken up by the carrier complex responsible for the uptake of taurocholate in the hepatocytes. Both the acute toxicity to D. magna and cytotoxicity of MX and MCA was rather low (EC₅₀ values > 0.1 mM) and we conclude that it is very unlikely that MX and MCA at concentrations occurring in recipients receiving chlorination effluents from pulp mills or chlorinated domestic sewage, as regards their acute toxicity, implies a risk for aquatic animals.     

Toxicity of selenate and selenite to the potworm <Emphasis Type="Italic">Enchytraeus albidus</Emphasis> (Annelida: Enchytraeidae): a laboratory test

Little information is available about the toxicity of inorganic selenium forms in soil animals. Therefore, the effects of selenate and selenite on the mortality and reproduction of Enchytraeus albidus were examined in standard laboratory tests with chronic exposure. Total and available amount of selenate and selenite were tested in a calcareous loamy chernozem soil. The LC₅₀ of selenate was 5.69 (2.7–8.12) mg kg^-1 dry wt. for total Se and 4.74 (2.14–6.98) mg kg^-1 dry wt. for available Se. Selenite LC₅₀ was as high as 22.5 (19.6–25.7) mg kg^-1 dry wt. for total Se and 8.10 (6.8–9.6) mg kg^-1 dry wt. for available Se. The EC₅₀ of selenate was 0.41 (0.35–0.48) mg kg^-1 dry wt. for total Se and 0.28 (0.24–0.34) mg kg^-1 dry wt. for available Se. Selenite EC₅₀ was as high as 7.3 (6.2–8.5) mg kg^-1 dry wt. for total Se and 2.46 (2.05–2.91) mg kg^-1 dry wt. for available Se. The response in reproduction was more sensitive to Se toxicity than the response in mortality. Selenate proved to be more toxic than selenite. Available data show that E. albidus may function as a biological indicator for some inorganic selenium forms in the soil.     

Embryotoxic and teratogenic effects of selenium in the diet of mallards

Mallards (Anas platyrhynchos) were fed a control diet, diets containing 1, 5, 10, or 25 ppm Se as sodium selenite, or a diet containing 10 ppm Se as seleno-DL-methionine in the first of two experiments. Selenium at 10 ppm as selenomethionine or 25 ppm as sodium selenite caused a 40-44% decrease in the total number of eggs that hatched compared to controls. Selenium at 25 ppm (sodium selenite) resulted in a 19% decrease in mean embryonic weight at 18 d of incubation, accompanied by a 6% decrease in crown-rump length. Ten parts per million Se as selenomethionine was more teratogenic than sodium selenite at 25 ppm. Selenomethionine (10 ppm Se) resulted in an incidence of 13.1% malformations that were often multiple, whereas sodium selenite (10 and 25 ppm Se) resulted in 3.6 and 4.2% malformations. The teratogenicity of selenomethionine was confirmed in a second experiment in which mallards received 1, 2, 4, 8, or 16 ppm Se as selenomethionine, resulting in 0.9, 0.5, 1.4, 6.8, and 67.9% malformations, respectively. These malformations included hydrocephaly, microphthalmia, lower bill defects, and foot defects with ectrodactyly. Both forms of selenium increased the incidence of edema and stunted embryonic growth. Selenomethionine (10 ppm Se) resulted in a significant increase of approximately 40% in plasma glutathione peroxidase activity and a 70% increase in sorbitol dehydrogenase activity (indicative of hepatotoxicity) in hatchlings. Sodium selenite (25 ppm Se) resulted in fourfold elevation in plasma uric acid concentration, indicative of renal alteration. Selenomethionine accumulated much better in eggs than did sodium selenite. These findings indicate that selenomethionine is considerably more teratogenic and generally more embryotoxic than sodium selenite, probably due to higher uptake of selenomethionine.