Helisoma Duryi Research Articles

Evaluation of the teratogenic potency of bulk zinc oxide and its nanoparticles on embryos of the freshwater snail, Helisoma duryi

Bulk zinc oxide (ZnO-BPs) and its nanoparticles (ZnO-NPs) are frequently used in various products for humans. Helisoma duryi embryos can serve as effective model organisms for studying the toxicity of NPs. This study aimed to compare the teratogenic potency of ZnO-BPs and ZnO NPs in the embryonic stages of H. duryi to evaluate the utility of this snail as a bioindicator for ZnO-NPs in the aquatic environment. The mechanisms of teratogenesis were evaluated by determination of the LC50, studying the effect of sub-lethal concentrations of both ZnO forms on the embryos, and studying their enzyme activity, oxidative stress, and biochemical analysis. The SDS-PAGE electrophoresis was undertaken to assess the effect of ZnO-BPs and ZnO NPs on protein synthesis. The results revealed that the veliger stage of H. duryi is the specific stage for bulk and nano ZnO. ZnO-NPs proved to be more toxic to snails’ embryos than ZnO-BPs. Exposure to ZnO influences specific types of defects in development, which in the case of BPs are far less drastic than those caused by NPs. Thus, the toxicity of ZnO-NPs in embryonic development is due to their unique physicochemical properties. The observed malformations include mainly hydropic malformation, exogastrulation, monophthalmia, shell misshapen, and cell lyses. Almost all tested oxidative biomarkers significantly changed, revealing that ZnONPs display more oxidative stress than ZnO-BPs. Also, the low concentration of ZnO induces many disturbances in the organic substances of veliger larvae, such as a decrease in the total protein and total lipid levels and an increase in the glycogen level. The results indicated that ZnO-BPs increase the number of protein bands. Conversely, ZnO-NPs concealed one band from treated egg masses, which was found in the control group. Embryos of snail are an appropriate model to control freshwater snails. This study demonstrates that H. duryi embryos can serve as effective model organisms to study the toxicity of ZnO-NPs.

Reproductive performance of freshwater snail, Helisoma duryi under the effect of bulk and nano zinc oxide.

Nanotechnology has been used to apply nanoparticle essential elements to enhance the ability of animals to absorb these elements and consequently improve their reproductive performance. High concentrations of nanoparticles (NPs) can directly harm a range of aquatic life forms, ultimately contributing to a decline in biodiversity. Helisoma duryi snails are a good model for studying the toxicological effects of bulk zinc oxide (ZnO-BPs) and nano zinc oxide (ZnO-NPs) on freshwater gastropods. This study aimed to compare the toxic effects of ZnO-BPs and ZnO-NPs on H. duryi snails and explore how waterborne and dietary exposure influenced the reproductive performance of this snail. ZnO-BPs and ZnO-NPs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and X-ray powder (XRD). This study revealed that the size of ZnO-BPs and ZnO-NPs were 154 nm and 11-31 nm, respectively. The results showed that exposure of adult snails to sub-lethal concentrations of both ZnO forms (bulk and nano) for 24 h/week for 4 weeks markedly changed their reproductive performance in a concentration-dependent manner, where fecundity was negatively affected by high concentrations. It was concluded that dietary exposure to the lowest tested concentration of ZnO-NPs (1 ppm) has a positive effect as the number of eggs and egg masses/snails increased and the incubation period decreased. Also, poly-vitelline eggs (The formation of twins) were observed. ZnO-NPs at low concentrations positively affect the reproductive performance of snails, especially after dietary exposure. The results revealed that 1 ppm ZnO-NPs could be supplementary provided to snails to improve their fertility, reduce the developmental time course, increase hatchability percentage, and produce poly-vitelline eggs.

Mycofiltration of Aqueous Iron (III) and Imidacloprid Solutions, and the Effects of the Filtrates on Selected Biomarkers of the Freshwater Snail Helisoma duryi

To alleviate the burden of water contamination, a newly developed form of bioremediation known as mycofiltration can be employed. Mycofiltration is an environment-friendly technology involving the treatment of contaminated water by passing it through a network of saprophytic fungal mycelium. A mycofilter made of Pleurotus ostreatus was used for the removal of iron (III) and imidacloprid from aqueous solutions. Batch mycofiltration, at a dosage of 1 g of mycofilter per 50 mL, was performed on iron (III) solutions of different concentrations (0.99, 10.7, 22.9, and 27.72 mg/L) and pH (3.3, 7 and 11). For column mycofiltration, the mycofilter was packed into pyrex columns (3.3 × 15 cm) to desired bed heights. Iron (III) and imidacloprid solutions of 18.99 mg/L and 234.70 ng/L, respectively, were filtered at a constant flow rate. Thereafter, Helisoma duryi snails were exposed for 96 h to the respective filtrates, and their catalase and acetylcholinesterase activities were assessed. Batch mycofiltration showed iron (III) removal rates as high as 85%. Column mycofiltration showed removal rates of 94 and 31% for iron (III) and imidacloprid, respectively. Catalase activity was significantly reduced (p < 0.05) in the snails exposed to iron (III) or imidacloprid filtrates, compared to the snails exposed to the non-mycofiltered media. A significantly higher acetylcholinesterase activity was induced by iron (III) filtrates in comparison with the non-mycofiltered media (p < 0.05). There were no significant differences in acetylcholinesterase activity (p > 0.05) in the snails exposed to mycofiltered and non-mycofiltered imidacloprid media. Mycofilter characterisation using Fourier Transform Infrared Spectrophotometry revealed significant changes in transmittance intensity in the mycofilters used for the iron (III) vs the ones used for the imidacloprid solutions. Mycofiltration was found to improve water quality although iron (III) was removed more effectively than imidacloprid.

Biomonitoring of manganese metal pollution in water and its impacts on biological activities of Biomphalaria alexandrina snail and larvicidal potencies

Metal pollution has many dangerous environmental and human health consequences due to the bioaccumulation in the tissues. The present study aims to measure the bioaccumulation factor of the manganese (Mn) heavy metal in Biomphalaria alexandrina snails’ tissues and water samples. The current results showed the concentration of Mn heavy metal in water (87.5 mg/l) and its bioaccumulation factor in Helisoma duryi tissue was higher than that in tissues of Physa acuta and B. alexandrina snails. Results showed that 87.5 mg/l Mn concentration had miracidicidal and cercaricidal activities. Also, this concentration decreased the mean total number of the hemocytes after exposure for 24 h or 48 h, while increasing both the mean mortality and phagocytic indices of the hemocytes of exposed snails. It caused alterations in the cytomorphology of the hemocytes of exposed snails after 24 or 48 h, where the granulocytes had irregular cell membranes and formed pseudopodia. Besides, levels of testosterone (T) and estradiol (E) were increased after exposure to 87.5 mg/l Mn metal compared to the control group. Also, it increased MDA (malonaldehyde) and TAC (total antioxidant capacity) contents, while decreasing SOD (superoxide dismutase). Besides, it caused significant histopathological damages in both hermaphrodite and digestive glands, represented in the degeneration of the gonadal, digestive, secretory cells, and the connective tissues. Therefore, B. alexandrina might be used as a sensitive bioindicator of pollution with Mn heavy metal to avoid ethics rules; besides, they are readily available and large in number.

An assessment of toxic effects of chemical toxicants on biochemical markers in the freshwater snail Helisoma duryi exposed to different water samples from a gold mine

Mining operations produce large amounts of waste effluent that contain compounds of varying toxicity that leach into aquatic bodies. We investigated the effects of water samples from a gold mining area on oxidative stress biomarkers in Helisoma duryi. Water was collected from four sites in the mining area: site 1, Mtshabezi water from a river 3 km from the mine and sites 2, 3 and 4 (underground water, waste effluent, process water) at the mine. The water samples were analysed for cyanide and heavy metals. Snails in groups of 30 were exposed to one liter volumes of water from sites 1- 4 and the control for up to 14 days. Cyanide levels in the 0.1 – 0.5 mg/L range were observed in water sampled from sites 1- 4. Cadmium, chromium, copper, lead and zinc were observed in all water samples from the mining area. Superoxide dismutase and catalase activities were enhanced in snails exposed to samples from the mining area. Increased concentrations of malondialdehyde, which indicates oxidative stress were observed in snails exposed to water from sites 1-4, suggesting that mining operations directly or indirectly release pollutants that include oxidative stressors which affect the wellbeing of aquatic organisms.

Assessment of schistosomiasis transmission in the River Nile at Greater Cairo using malacological surveys and cercariometry.

Continuous field studies on the abundance and distribution of freshwater snails and cercarial populations are important for schistosomiasis control programs. In the present work, snail surveys and cercariometry were conducted for four successive seasons at 12 sites on the Nile River banks in the area of Greater Cairo to identify potential transmission foci for schistosomiasis. In addition, water physicochemical parameters were recorded. The results showed that the electrical conductivity, total dissolved solids, dissolved oxygen, and pH were within the permissible levels, except that the water temperature increased, especially in the spring season. Malacological surveys identified 10 native snail species at the studied sites of the Nile River, namelyBulinus truncatus, Biomphalaria alexandrina, Lymnaea natalensis, Lanistes carinatus, Cleopatra bulimoides, Melanoides tuberculata, Helisoma duryi, Bellamya unicolor, Physa acuta, Thedoxus niloticus,andone invasive snail species,Thiara scabra. The calculated diversity index indicated that the structure of snails' habitats was poor, while Evenness index indicated that the individuals were not distributed equally. Natural infection results identified no schistosome cercariae inB. truncatusandB. alexandrina. However, the cercariometry recoveredSchistosomacercariae in all the surveyed sites during all seasons with variable distribution. The preceding data suggest that there are still some active transmission foci for schistosomiasis infection in the Nile River. Moreover, the present finding highlights the importance of cercariomety as a complementary approach to snail samplings for identifying the transmission foci for schistosomiasis.

Embryonic Development of the Ramshorn Snail Helisoma duryi (Wetherby 1879) (Gastropoda, Planorbidae) from Gharbia Province, Egypt

Embryonic development of the freshwater planorbid snail, Helisoma duryi, reared under laboratory controlled conditions was described in detail. The morphological characters and the time course of embryonic stages are presented. H. duryi generally lay egg masses at night or in the early morning hours. The egg type of H. duryi is isolecithal, and the cleavage is spiral holoblastic. After early cleavages followed by the appearance of the blastula, gastrula, and larval (trochophore, veliger, veliconch) stages, the fully developed young snail start hatched on the eighth day. Many unique developmental features in studied snail were observed and discussed.

Effect of Bisphenol A (EDC) on the reproductive potential of Helisoma duryi (Wetherby, 1879)

It has been claimed that bisphenol A (BPA) induces some reproductive potential disorders in the freshwater gastropods worldwide. The adult fecundity and egg hatchability as well as histological effects and embryonic deformities on the exotic freshwater snail Helisoma duryi have been studied to explore the superfeminization phenomena. The results of the 96h.LC50 of BPA for H. duryi was 4.06, and the histological examination for the hermaphrodite gland which was exposed to sub-lethal concentration showed considerable modification and changes in the semineferoustubules, number of eggs, spermatogenesis and the thickness of surrounding connective tissues. The results highlight the importance of obtaining basic knowledge of information on sub-lethal damage through the life-history of potential test species after exposure to test protocols.

The molluscicidal efficacy of methanol extracts of Pterocarpus angolensis, Sclerocarya birrea, Pappea capensis and Commiphora africana against Lymnaea natalensis and Helisoma duryi

Tropical plants have been shown to contain compounds that have some molluscicidal activity against snail intermediate hosts of parasitic trematodes. Dried methanol extracts of Pterocarpus angolensis, Sclerocarya birrea, Pappea capensis and Commiphora africana were screened for molluscicidal activity against Lymnaea natalensis and Helisoma duryi. The freshwater snail L. natalensis is the intermediate host of fascioliasis in Zimbabwe, whereas H. duryi is a related non-vector snail. Of the four plant extracts studied, P. angolensis was the most effective with a 48 h LC50 of 34 mg l−1, whilst C. africana was the least effective with a 48 h LC50 of 53 mg l−1 for L. natalensis. The extract of P. angolensis was selectively toxic to L. natalensis, its LC50 being three times higher for H. duryi than for L. natalensis. These plant extracts are a potential source of lead compounds in the development of highly selective and environmentally friendly molluscicides. Further studies to isolate the active principles in these extracts are recommended.

Pollutants, Snails, Oxidative-Stress, Organophosphates, Metals

Aquatic reservoirs remain the ultimate sink of chemical pollutants emanating from anthropogenic activities such as agriculture, mining and industry. Freshwater biota undoubtedly is at risk from the adverse effects of these water pollutants and there is therefore, a need to monitor effects of these chemical pollutants in order to safeguard the health of aquatic biota. We investigated the oxidative stress effects of chlorpyrifos and lead on the freshwater snail Helisoma duryi to assess the potential of using this enzyme system as a biondicator of exposure to environmental pollutants. Groups of snails were exposed to 5 ppb lead acetate and 25 ppb chlorpyrifos for 7 days after which half of the snails were sacrificed and the other half were allowed to recover in clean water and sacrificed after another 7 days. Post mitochondrial fractions were used to measure the activities of the following antioxidant enzymes: superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase and diphosphotriphosphodiaphorase. Both pollutants enhanced the activities of all the antioxidant enzymes suggesting a defensive mechanism by the snail to combat the oxidative stress due to the organophosphate chlopryrifos and metal pollutant lead. There was a significant recovery of the antioxidant defense system of the snails allowed to recover in clean water shown by the reduced alteration of the antioxidant enzyme activities of the snails allowed to recover for 7 days. This suggests the need to minimize exposure of aquatic biota to chemical pollutants and remediate the polluted water reservoirs in order to safeguard the health of aquatic life.

English

This study involves an investigation on the effects of a pesticide, metal and a detergent as individual and mixtures on esterase and antioxidant enzyme activity of the freshwater snail Helisoma duryi. Adult snails were exposed to sublethal concentrations of copper (5 &micro;g/L), industrial detergent, oxyfoam (15 &micro;g/L), carbaryl (25 &micro;g/L) as well as mixtures of these pollutants for 96 hours. Carboxylesterase and cholinesterase activities were measured using 4-nitrophenyl acetate and acetylthiocholine iodide as substrates respectively. The activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione S- transferase were also measured as indices of oxidative stress. Esterase activity was inhibited in snails exposed to carbaryl, copper or detergent. Mixtures of the different chemicals also caused inhibitions of esterase activity when compared to the controls. &nbsp;All the chemicals individually and as mixtures, caused enhanced activities of antioxidant enzymes. When comparing all the antioxidant enzymes analyzed, the highest activity was caused by the triple mixture of the pollutants. The results suggest that aquatic life is at risk to adverse effects of pollutant mixture as reflected by increased alteration of enzyme activity in mixture- exposed snails though the increase was less than sum of effects of individual pollutants. More studies on the effects of a wider range of pollutant mixtures on aquatic organisms are needed however, for the full appreciation of reactive interactions that takes place in complex mixtures which ultimately affect the health of aquatic biota. &nbsp; Key words: Pollutants, snails, oxidative stress, esterases, detergents.

Fresh water snails as bioindicator for some heavy metals in the aquatic environment

AbstractThe aim of this investigation is to study some freshwater snails as bioindicator for heavy metals Cd, Cu and Pb by determining the concentration of these metals in the field water samples and in whole snail tissues. Seven freshwater snails were used in the present study, some of which are considered medically important snails in Egypt, Biomphalaria alexandrina and Bulinus truncatus, the intermediate hosts for schistosomiasis and nontarget snails Bellamya unicolor, Cleopatra bulimoides, Helisoma duryi, Physa acuta and Theodoxus niloticus. Samples of snails were gathered from three Egyptian governorates: Damietta, Giza and Monufia.. The snails were arranged according to their accumulated concentration of the above‐mentioned microelements in descending order as follows: C. bulimoides &gt; H. duryi &gt; B. truncatus &gt; B. alexandrina &gt;P. acuta &gt; B. unicolor &gt; T. niloticus. It is concluded from the analysis of water and the investigated snails that these snails can accumulate Cu, Pb and Cd with high concentrations in their bodies, so they can be used as bioindicators for heavy metals.

Community structure of molluscans in River Nile and its branches in Assiut governorate, Egypt

The present work aims to survey the benthic mollusc communities in River Nile and its branches in Assiut governorate, Egypt. Twenty-six species were recorded from the collecting sites during the period from March 2010 until March 2012. These species belong to fifteen families; eleven families of Gastropoda and four families of Bivalvia. The constant species in this survey were five species: Cleopatra bulimoides, Lanistes varicus, Lanistes carinatus, Melanoides tuberculata and Caelatura aegyptiaca. The accessory species were seventeen species: Mutela rostrata, Pila wernei, Sphaerium sp., Pila ovata, Bithynia connollyi, Lymnaea natalensis, Corbicula fluminalis, Theodoxus niloticus, Unio teretiusculus, Bellamya unicolor, Hydrobia aponensis, Pseudosuccinea columella, Valvata nilotica, Biomphalaria alexandrina, Bulinus truncates, Physa acuta and Gabbiella senaariensis and the accidental species were four species: Helisoma duryi, Succinea cleopatra, Corbicula fluminea and Gyraulus ehrenbergi. The species richness and diversity of molluscan community reach the maximum level in spring and summer months.

Population dynamics of freshwater snails (Mollusca: Gastropoda) at Qena Governorate, Upper Egypt

Monthly and seasonal fluctuations in densities of freshwater snails were recorded in six different sites at Qena Governorate during the period of March 2009 February 2010. The study revealed the occurrence of 13 species of freshwater snails belonging to 9 families of class Gastropoda. The snail species and their total catch were: Cleopatra bulimoides (1704 specimens), Lanistes carinatus (1468 specimens), Melanoides tuberculata (1036 specimens), Gabbiella senaariensis (731 specimens) Theodoxus niloticus (670 specimens), Bellamya unicolor (588 specimens), Physa acuta (213 specimens), Lymnaea natalensis (145 specimens), Succinea cleopatra (85 specimens), Bulinus truncatus (73 specimens), Biomphalaria alexandrina (41 specimens), Gyraulus ehrenbergi (12 specimens) and Helisoma duryi (2 specimens) in a descending order. The densities of the recorded snails varied seasonally and the general seasonal peak was recorded during Autumn while the lowest density observed during Winter. Ecological factors which may influence the distribution of the recorded snails were also determined (water temperature, pH, dissolved oxygen conductivity, total hardness, total dissolved salts, water depth and percentage of macrophytes).

Complex intestinal bacterial communities in three species of planorbid snails

The intestinal microbiota of animals play an important role in their energy metabolism and resistance to pathogens, and thus in maintenance of animals in their environment. We surveyed the intestinal microbiota of three species of Planorbidae (Gastropoda: Pulmonata). Cultivation-independent molecular methods were used to investigate the community composition of intestinal bacteria from individual snails: three Biomphalaria pfeifferi (Africa), two Bulinus africanus (Africa) and three Helisoma duryi (North America). PCR amplification and sequencing of 612 bacterial 16S rRNA genes yielded 282 unique DNA sequences (97% sequence similarity) among the eight individual snails sampled. The observed bacterial diversity was distributed over 18 phyla, with the greatest number of 16S rRNA gene sequences derived from the Gammaproteobacteria, Bacteroidetes and Acidobacteria. These results document the presence of highly diverse gut bacterial communities in three snail species and indicate the need for additional study to determine the roles that gut microbes play in the physiology and immunology of planorbid snails.

Biological, biochemical, and molecular parameters of Helisoma duryi snails exposed to the pesticides Malathion and Deltamethrin

Aquatic environments get contaminated with pesticides residues that result from the application of pesticides in agricultural practices. The present study aimed to evaluate the pesticides Malathion and Deltamethrin on biological and biochemical parameters of Helisoma duryi snails. The results showed that LC 10 of the two pesticides caused considerable reduction in survival rates and egg production of treated snails. Glucose concentration in hemolymph of the exposed snails showed a significant increase. On the other hand, albumin in hemolymph and glycogen contents as well as the activities of enzymes in tissues of snails including lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), arginase, and ornithine aminotransferase (OAT) were significantly inhibited. Meanwhile, the activity of AMP deaminase of the exposed snails was significantly increased in the hemolymph in response to treatment. The electrophoretic pattern of total protein showed differences in number and molecular weights of protein bands. DNA concentration was investigated by measuring the intensity of the genomic bands and its showed its increase in the treated snails. It was concluded that the residues of Malathion and Deltamethrin pesticides in aquatic environments have toxic effects on non-target organism, e.g. H. duryi snails.

Incompatibility Between Miracidia of Schistosoma mansoni and Helisoma duryi Occurs at Two Stages: Penetration and Intramolluscan Establishment

Helisoma spp. snails are not susceptible to infection with miracidia of Schistosoma mansoni because the miracidia do not penetrate them. However, in view of the phylogenetic proximity and histocompatibility between Helisoma spp. and the normal intermediate host, Biomphalaria glabrata , schistosome miracidia conceivably could survive if experimentally introduced into the hemocoel of Helisoma spp. To test this hypothesis, schistosome-susceptible NIH albino B. glabrata, schistosome-resistant Salvador B. glabrata, and Helisoma duryi were injected with miracidia of S. mansoni, and the outcome was followed both by monitoring snails for infection for several weeks and by histological examination at 24 and 48 hr post-injection (PI). Patent infections developed in most NIH albino snails but in none of the Salvador B. glabrata or H. duryi individuals. Histological analysis showed a higher proportion of normal sporocysts in various tissues of NIH albino snails at both time periods relative to Salvador snails, which contained mostly sporocysts undergoing hemocytic encapsulation. In H. duryi , nearly all sporocysts were dead by 48 hr PI.

Enzyme analyses on seven laboratory stocks and two natural populations of Helisoma duryi. Electrophoretic patterns of eight enzymes with genetic information on four polymorphic enzymes

The relative mobility values observed in 8 enzyme systems were recorded from 9 stocks of Helisoma duryi. The enzymes phosphoglucose isomerase, hydroxybutyrate dehydrogenase, esterase-A and ester-ase-d showed polymorphisms. A genetic analysis of the polymorphic enzymes revealed autosomal, codominant inheritance of the different morphs. No linkage has been detected between the enzyme loci and the albino gene. In a comparison with African members of the genus Biomphalaria it is suggested that the enzymes Hbdh and Pgm be used for a reliable identification.

Potential marker enzymes and metal–metal interactions in Helisoma duryi and Lymnaea natalensis exposed to cadmium

In this study, we investigate the effects of exposure to cadmium and copper on Lymnaea natalensis and Helisoma duryi. The snails were dosed with Cd 2+ or Cu 2+ for a period of 96 h. Snails dosed with Cd accumulated the metal significantly ( P<0.05) in tissues but not in shells. Mortality was observed at approximately 1 mg Cd/l of culture water. In tissues and shells of snails dosed with Cd or Cu, synergistic and antagonistic metal–metal interactions involving Cd, Cu, Zn, and Pb were observed and these may affect metal toxicity. Glutamate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase were assayed in whole snail tissue sub-cellular fractions of Cd-dosed snails. Generally, enzyme activity significantly increased at lower concentrations of Cd but decreased at high concentrations of the metal. However, mitochondrial alanine aminotransferase activity progressively declined with increasing Cd concentration. The changes in some of the enzymes’ activities suggest biomarker potential.

Comparative studies on the uptake and effects of cadmium and zinc on the cellular energy allocation of two freshwater gastropods

The uptake and effects of cadmium (Cd) and zinc (Zn) as free metal ions were compared in the freshwater gastropods Melanoides tuberculata and Helisoma duryi. The Langmuir isotherm model was applied to determine the uptake of Cd, Zn, and a mixture of the metals at five different concentrations (Cd: 0.25, 0.51, 0.77, 1.03, 1.29 μM; Zn: 0.17, 0.43, 0.86, 2.17, 4.34 μM). The model gave a good description of metal uptake over a short exposure period (6 h). The gastropods showed interspecies differences in the uptake of Zn. The linear uptake of Cd was similar in these species, although the data did not yield a good fit to the model. No clear Cd/Zn interaction was observed with the mixed metal exposures and both species showed a reduced net uptake for the metals. Cellular energy allocation as a biomarker of exposure provided a measure of the net energy budget (total available energy reserves and total energy consumption). The gastropods were exposed to 0.51 μM Cd, 0.43 μM Zn, and mixture of the metals for a 2-week period. Both species elicited similar decreased net energy budgets following the metal exposures. A combined study on metal uptake and biomarker responses in organisms allows the ability to make interspecies sensitivity comparisons in ecotoxicological studies.