A new simple method with high precision for determining the toxicity of antifouling paints on brine shrimp larvae (Artemia): first results

The use of antifouling paints is the only truly effective method for the protection of underwater structures from the development of fouling organisms. In the present study, the surface to volume concept constitutes the basis for the development of a new and improved method for determining the toxicity of antifouling paints on marine organisms. Particular emphasis is placed on the attainment of a standardized uniformity of coated surfaces. Failure to control the thickness of the coat of paint in previous studies of this type, has led to inaccurate evaluation of the relative toxicity of samples. Herein, an attempt is made to solve this problem using a simple technique which gives completely uniform and smooth surfaces. The effectiveness of this technique is assessed through two series of experiments using two different types of test containers: 50 ml modified syringes and 7 ml multiwells. The results of the toxicity experiments follow a normal distribution around the average value which allows to consider these values as reliable for comparison of the level of toxic effect detected with the two types of test containers. The mean lethal concentration L(S/V)(50) in the test series conducted in the multiwells (20.38 mm(2)ml(-1)) does not differ significantly from that obtained in the test series using modified syringes (20.065 mm(2)ml(-1)). It can thus be concluded from this preliminary study that the new method and the two different ways of exposing the test organisms to the antifouling paints and their leachates gave reliable and replicable results.     

Trophic transfer of paralytic shellfish toxins from clams (Ruditapes philippinarum) to gastropods (Nassarius festivus)

A local strain of the dinoflagellate Alexandrium tamarense (ATCI01), which predominantly produces C2 toxin, was fed to the clams (Ruditapes philippinarum) under laboratory conditions. Concentrations of paralytic shellfish toxins (PSTs) in the dosed clams were determined by High Performance Liquid Chromatographic (HPLC) analyses, and the clams were homogenized and then fed to the gastropods (Nassarius festivus). In the toxin accumulation phase, which lasted for 42 days, concentrations of PSTs increased in the snails gradually, reaching a maximum of 1.10 nmole g(-1) at the end of the exposure period. The toxin content of the homogenized clams (food) was 13.18 nmole g(-1), which was about 12-fold higher than the PST content in the snails. Between day 43 and day 82, the snails were fed with non-toxic clams, and this period represented the depuration phase. Accumulation and depuration rates of PSTs in the snails, N. festivus, were determined by fitting the experimental data to user-defined parameters program using a one-compartment model. Two different modeling approaches were used to derive the accumulation and depuration rates. The first approach is to derive both values from the data for the toxin uptake. The second approach is to derive depuration rate from the depuration data and then to derive uptake rate, allowing for toxin depuration, from the data for toxin uptake. The first approach yielded more consistent results for the toxin concentration at the end of the uptake period, when compared with the experimental data. The toxin uptake and depuration rates were 1.64 (pmole of toxin into snail per day) per (nmole g(-1) of toxin in food) and 0.06+/-0.02 day(-1) (mean+/-SE), respectively. The toxin profiles of snails were similar to the clams, but different from the algae. Besides C toxins (C1 and C2), dcGTX2 and dcGTX3 were also detected in both clams and snails. The beta:alpha epimer ratio gradually decreased during trophic transfer and approached a ratio of 1:3 (26.4 mol%:73.6 mol% at day 42) in the snails, near the end of the accumulation period.     

Trophic transfer of pyrene metabolites and nonextractable fraction from Oligochaete (Lumbriculus variegatus) to juvenile brown trout (Salmo trutta)

Cell lines of Etroplus suratensis established in our laboratory were evaluated for their potential use as screening tools for the ecotoxicological assessment of tannery effluent. The cytotoxic effect of tannery effluent in three cell lines derived from eye, kidney and gill tissue of E. suratensis was assessed using multiple endpoints such as Neutral Red (NR) assay, Coomassie Blue (CB) protein assay and Alamar Blue (AB) assay. Acute toxicity tests on fish were conducted by exposing E. suratensis for 96 h to tannery effluent under static conditions. The toxic effect of tannery effluent on the survival of fish was found to be concentration and time dependent. The tannery effluent at the concentration of 15% caused 100% mortality at 96 h whereas the lower concentration (0.5%) caused 13.33% mortality. The cytotoxicity of tannery effluent was found to be similar in the three cell lines tested, independent of the toxic endpoints employed. EC₅₀ values, the effective concentration of tannery effluent resulting in 50% inhibition of cytotoxicity parameters after 48 h exposure to tannery effluent were calculated for eye, kidney and gill cell lines using NR uptake, AB and cell protein assays. Statistical analysis revealed good correlation with r² = 0.95-0.99 for all combinations between endpoints employed. Linear correlations between each in vitro EC₅₀ and the in vivo LC₅₀ data, were highly significant p < 0.001 with r² = 0.977, 0.968 and 0.906 for AB₅₀, NR₅₀, and CB₅₀, respectively.     

Trophic transfer and accumulation of mercury in ray species in coastal waters affected by historic mercury mining (Gulf of Trieste,northern Adriatic Sea)

Total mercury (Hg) and monomethylmercury (MMHg) were analysed in the gills, liver and muscle of four cartilaginous fish species (top predators), namely, the eagle ray (Myliobatis aquila), the bull ray (Pteromylaeus bovinus), the pelagic stingray (Dasyatis violacea) and the common stingray (Dasyatis pastinaca), collected in the Gulf of Trieste, one of the most Hg-polluted areas in the Mediterranean and worldwide due to past mining activity in Idrija (West Slovenia). The highest Hg and MMHg concentrations expressed on a dry weight (d.w.) basis were found in the muscle of the pelagic stingray (mean, 2.529 mg/kg; range, 1.179–4.398 mg/kg, d.w.), followed by the bull ray (mean, 1.582 mg/kg; range, 0.129–3.050 mg/kg d.w.) and the eagle ray (mean, 0.222 mg/kg; range, 0.070–0.467 mg/kg, d.w.). Only one specimen of the common stingray was analysed, with a mean value in the muscle of 1.596 mg/kg, d.w. Hg and MMHg contents in the bull ray were found to be positively correlated with species length and weight. The highest MMHg accumulation was found in muscle tissue. Hg and MMHg were also found in two embryos of a bull ray, indicating Hg transfer from the mother during pregnancy. The number of specimens and the size coverage of the bull rays allowed an assessment of Hg accumulation with age. It was shown that in bigger bull ray specimens, the high uptake of inorganic Hg in the liver and the slower MMHg increase in the muscle were most probably due to the demethylation of MMHg in the liver. The highest Hg and MMHg contents in all organs were found in the pelagic stingray, which first appeared in the northern Adriatic in 1999. High Hg and MMHg concentrations were also found in prey species such as the banded murex (Hexaplex trunculus), the principal prey of the eagle rays and bull rays, the anchovy (Engraulis encrasicholus) and the red bandfish (Cepola rubescens), which are preyed upon by the pelagic stingray, as well as in zooplankton and seawater. Based on previously published data, a tentative estimation of MMHg bioamagnification was established. The average increase in MMHg between seawater, including phytoplankton, and zooplankton in the Gulf was about 10⁴, and MMHg in anchovy was about 50-fold higher than in zooplankton. The bioaccumulation of MMHg between seawater and small pelagic fish (anchovy) amounted to 10⁶ and between water and the muscle of larger pelagic fish (pelagic stingray) to 10⁷. The MMHg increase between surface sediment and benthic invertebrates (murex) and between benthic invertebrates and small benthic fish was 10². Ultimately, the trophic transfer resulted in a 10³ accumulation of MMHg between water and muscle of larger benthic fish (bull ray, eagle ray, common stingray), suggesting lower bioaccumulation by benthic feeding species.     

Electrospray Ionization Mass Spectrometry (ESI-MS) monitoring of the photolysis of diazinon in aqueous solution: Degradation route and toxicity of by-products against Artemia salina

The photolytic degradation of diazinon, an organophosphorus pesticide, in aqueous medium under assorted pH values was continuously monitored by direct infusion electrospray ionization mass spectrometry (ESI-MS). The results indicated that the UV radiation was quite efficient in promoting the pesticide degradation at the three pH levels evaluated (5, 7 and 8). The m/z of the most abundant ions observed in the mass spectra (MS), in conjunction with the fragmentation patterns of such ionic species (MS/MS data), made possible the proposition of chemical structures for the main by-products formed. As a result, routes for the photodegradation of diazinon in aqueous solution could thus be suggested. In the assays using Artemia salina (brine shrimp) it was verified that the photodegradation products exhibited much lower toxicity than the primary substrate. Aiming at mimicking the conditions ordinarily found in water treatment plants, an additional series of tests was conducted with a solution containing sodium hypochlorite and diazinon. This solution, when not exposed to UV radiation, exhibited high toxicity against the microorganisms. Under the influence of UV radiation, however, the toxicity rates decreased dramatically. This result is relevant because it points toward the confident application of UV radiation to neutralize the deleterious effects caused by diazinon (and perhaps other organophosphorus pesticides) as well as sodium hypochlorite to the environment.     

An uptake and elimination kinetics approach to assess the bioavailability of chromium,copper, and arsenic to earthworms (Eisenia andrei) in contaminated field soils

The aim of this study was to determine the bioavailability of metals in field soils contaminated with chromated copper arsenate (CCA) mixtures. The uptake and elimination kinetics of chromium, copper, and arsenic were assessed in the earthworm Eisenia andrei exposed to soils from a gradient of CCA wood preservative contamination near Hartola, Finland. In soils contaminated with 1480–1590 mg Cr/kg dry soil, 642–791 mg Cu/kg dry soil, and 850–2810 mg Ag/kg dry soil, uptake and elimination kinetics patterns were similar for Cr and Cu. Both metals were rapidly taken up and rapidly excreted by Eisenia andrei with equilibrium reached within 1 day. The metalloid As, however, showed very slow uptake and elimination in the earthworms and body concentrations did not reach equilibrium within 21 days. Bioaccumulation factors (BAF) were low for Cu and Cr (< 0.1), but high for As at 0.54–1.8. The potential risk of CCA exposure for the terrestrial ecosystem therefore is mainly due to As.

     

Nickel and other metal uptake and accumulation by species of Alyssum (Brassicaceae) from the ultramafics of Iran

Some plants growing on serpentine (ultramafic) soils are able to hyperaccumulate nickel in their above-ground parts. The genus Alyssum L. contains the greatest number of Ni-hyperaccumulator plants so far reported. There are substantial areas of serpentine soils at many locations in Iran. This paper presents the analyses for Ni, Cr, Mn, Fe, Mg and Ca in soils and Alyssum species from the ultramafics of west and northwest Iran. Soil analysis for total elements in these areas indicates that typical concentrations of Ni, Cr, Mn, Fe, Mg and Ca are up to about 1240, 365, 800, 51,150, 152,390 and 11,790 microg g(-1), respectively. During this study, seven Alyssum species were collected. Analysis of leaf dry matter shows that Alyssum bracteatum can contain up to 2300 microg Nig(-1), while the other species contain much lower concentrations of Ni and other elements. A. bracteatum is endemic to Iran and the first Ni hyperaccumulator reported from this species.     

Tolerance to,and avoidance of,hypoxia by the penaeid shrimp (Metapenaeus ensis)

Aquatic hypoxia caused by eutrophication may lead to mass mortality of valuable living resources such as fish and shrimp. However, there is little information on the hypoxic tolerance of penaeid shrimp, and whether they are able to avoid hypoxia. In laboratory experiments, LC50, LT50 and heart beats per minute were determined for juvenile Metapenaeus ensis at 0.5, 1.0, 2.0 and 6.0 mg O2 l(-1). The 8-h LC50, for DO was 0.77 mg O2 l(-1), while the LT50 at 0.5 mg O2 l(-1) was 399 min. Heart beat rate significantly declined when DO fell below 1.0 mg O2 l(-1). When confronted with a gradient of dissolved oxygen, M. ensis were able to avoid hypoxic areas and move to oxygenated water. M. ensis appeared to be sensitive to hypoxia, and their ability to detect and avoid hypoxia may enhance their survival in habitats where hypoxia may occur.     

Exposure of Chironomus riparius larvae (diptera) to lead, mercury and beta-sitosterol: effects on mouthpart deformation and moulting

Mouthpart deformation in chironomid larvae is induced by exposure to chemical contaminants and is becoming an established bio-indicator in sediment assessment programmes. However, concentration-response relationships with causal agents have only been established occasionally and with varying success. In this laboratory study, instar II and III larvae were exposed to sub-lethal concentrations of lead, mercury and beta-sitosterol. A significant deformation response was induced in the pecten with lead and mercury. Deformation frequencies of the mentum after metal exposures were not significantly different from the control. Moulting was retarded by both metals and was well correlated with mouthpart deformation. The beta-sitosterol is an endocrine disruptor, which was used to test the hypothetical cause-effect relation between disruption of ecdyson functioning and chironomid deformation. In the present study, exposure to sublethal concentrations of beta-sitosterol did not result in any effect on deformation or moulting. As such, the proposed hypothesis of endocrine disruptors as primary causal agents of chironomid deformation could not be substantiated. Acetone, which was used as a solvent to apply beta-sitosterol caused a significant increase of mentum deformation. The ground filtration paper used as substrate seemed to induce deformities as well. Substrate contamination, acetone and (especially) inbreeding were most probably responsible for the high deformation frequencies in the control conditions.     

Tissue-specific accumulation and lactational transfer of polychlorinated biphenyls, chlorinated pesticides, and brominated flame retardants in hooded seals (Cistophora cristata) from the Gulf of St. Lawrence: applications for monitoring

Accumulation and mother-pup transfer of halogenated organic contaminants was studied in hooded seal tissues from eastern Canada. Blubber polychlorinated biphenyl (PCB) and total pesticide concentrations were relatively high, possibly due to their high trophic level and demersal feeding habits. Blood plasma showed the lowest contaminant concentrations compared to blubber and liver, possibly due to a lower affinity of these compounds to lipoproteins in blood plasma. Total contaminant body burden correlated well with blubber, liver, and milk contaminants, but not with blood plasma contaminants, indicating that blood plasma might be less suitable to monitor contaminants in hooded seals. Lactational transfer favored less lipophilic contaminants and was associated with relatively high blood plasma PCB and polybrominated diphenyl ether concentrations in females. Despite lactational transfer, females did not show significantly lower blubber contaminant concentrations or burdens than males. This might be caused by their low blubber, and thus contaminant, loss during lactation compared to other species.     

Rapid behavioural and morphological responses of hydropsychid larvae (Trichoptera, Hydropsychidae) to sublethal cadmium exposure

Larvae of Hydropsyche contubernalis and H. siltalai were exposed to sublethal cadmium concentrations of 0, 0.012, 0.16 and 10 mg/litre(-1) for 72 h. Linear logit models revealed a significant increase in the frequency and degree of damage of the anal papillae of both species with increasing Cd concentration. Hydropsyche contubernalis showed a stronger and earlier anal papillae response under Cd exposure than H. siltalai, whereas in the latter species darkening of the ventral sides of the abdomen was also observed. The lowest Cd level altered the competition behaviour of Hydropsyche contubernalis larvae after only 24 h exposure, yet no visual signs of morphological damage were detected. The exposure intruder larvae spent significantly less time trying to enter the nets of resident larvae than did unexposed intruders. In addition, both the exposed intruders and exposed residents pursued different behavioural tactics during the encounters compared to their unexposed counterparts.     

Heavy metal accumulation and histomorphological alterations in Aphanius fasciatus (Pisces,Cyprinodontidae) from the Gulf of Gabes (Tunisia)

The present study illustrates an analysis of histological changes; cadmium (Cd), copper (Cu) and zinc (Zn) accumulation; and metallothionein (MT) levels in normal and deformed Mediterranean killifish, Aphanius fasciatus (Pisces, Cyprinodontidae), collected from unpolluted (S1) and polluted areas (S2) in the Gulf of Gabes in Tunisia. Metal determination in water and sediment showed that the concentrations were significantly higher (p?Histopathological investigations revealed greater changes in gills, kidney, liver, and bone tissues of fish from the polluted area than those recorded in fish from the reference area. In comparison to normal fish of the polluted area (S2), tissue alterations were more developed in deformed specimens of this site. A possible relationship between metallic pollution, incidence of spinal deformities, and histological changes in A. fasciatus in the polluted site was discussed.     

The transfer of trichloroethylene (TCE) from a shower to indoor air: experimental measurements and their implications

Experiments were performed to measure the transfer of trichloroethylene (TCE), a volatile organic compound (VOC), from tap water in showers to indoor air. In these experiments, the loss of TCE from tap water in the shower is based on the difference between influent and effluent concentrations. We have developed and previously published a three-compartment model, which we use to simulate the 24-h concentration history of VOCs in the shower, bathroom, and remaining household volumes resulting from the use of contaminated tap water. An important input to this model is the transfer efficiency of the VOC from water to air. The experiments reveal that the transfer efficiency of TCE from shower water to air has an arithmetic mean value of 61 percent and an arithmetic standard deviation of 9 percent. Analysis of the results shows that there is no statistically significant difference between the transfer efficiency measured with hot (37 degrees C) or cold (22 degrees C) shower water and that there is no statistically significant change in transfer efficiency with time during a 20-min shower. The implications for exposure assessment are considered.     

The transfer of trichloroethylene (TCE) from a shower to indoor air: experimental measurements and their implications

Experiments were performed to measure the transfer of trichloroethylene (TCE), a volatile organic compound (VOC), from tap water in showers to indoor air. In these experiments, the loss of TCE from tap water in the shower is based on the difference between influent and effluent concentrations. We have developed and previously published a three-compartment model, which we use to simulate the 24-h concentration history of VOCs in the shower, bathroom, and remaining household volumes resulting from the use of contaminated tap water. An important input to this model is the transfer efficiency of the VOC from water to air. The experiments reveal that the transfer efficiency of TCE from shower water to air has an arithmetic mean value of 61 percent and an arithmetic standard deviation of 9 percent. Analysis of the results shows that there is no statistically significant difference between the transfer efficiency measured with hot (37 degrees C) or cold (22 degrees C) shower water and that there is no statistically significant change in transfer efficiency with time during a 20-min shower. The implications for exposure assessment are considered.     

Specific accumulation of arsenic compounds in green turtles (Chelonia mydas) and hawksbill turtles (Eretmochelys imbricata) from Ishigaki Island, Japan

Concentrations of total arsenic (As) and individual compounds were determined in green and hawksbill turtles from Ishigaki Island, Japan. In both species, total As concentrations were highest in muscle among the tissues. Arsenobetaine was a major compound in most tissues of both turtles. High concentrations of trimethylarsine oxide were detected in hawksbill turtles. A significant negative correlation between standard carapace length (SCL), an indicator of age, and total As levels in green turtles was found. In contrast, the levels increased with SCL of hawksbill turtles. Shifts in feeding habitats with growth may account for such a growth-dependent accumulation of As. Although concentrations of As in marine sponges, the major food of hawksbill turtles are not high compared to those in algae eaten by green turtles, As concentrations in hawksbill turtles were higher than those in green turtles, indicating that hawksbill turtles may have a specific accumulation mechanism for As.     

Dietary uptake of polybrominated diphenyl ethers (PBDEs), occurrence and profiles, in aquacultured turbots (Psetta maxima) from Galicia, Spain

Polybromodiphenyl ethers (PBDEs) are one of the many toxic chemicals present in the environment and in the food we eat every day, being fish one of the main sources of persistent organic pollutants in our diet; like other lipid-related contaminants, they are of concern since they can bioaccumulate and biomagnify through the trophic chain. We published a study focused on the dietary uptake of dioxins and furans (PCDD/Fs) and dioxin-like polychlorobiphenyls (dl-PCBs) in a set of samples of Spanish farmed turbot (Blanco et al., 2007). In the present paper, we extend the study to PBDEs to provide more information about the uptake and transfer from feed to fish of halogenated contaminants. PBDEs in the feeds (2.35-4.76 ng g(-1)) were reflected in turbot fillets (0.54-2.05 ng g(-1)): predominant congeners were tetra-BDE 47, penta-BDEs 99 and 100. It is remarkable that tetra-BDE 49, accounting for only 2% in the feed, contributed to 15% of total PBDEs in turbot fillets. Dietary net accumulation values, 30-45%, showed that tri-, tetra-, penta- and hexa-BDEs were as efficiently transferred into turbot as dl-PCBs and tetra- and penta-chlorinated PCDD/Fs. Lipid-normalized biomagnification factors relating concentration in fish and in feed, BMFs>1 were obtained, except for BDE 209. BDE 49 accumulation, 90%, was possibly contributed by metabolism of higher brominated BDEs. Implication in aquaculture management is a need for uncontaminated fish feed to offer safe products.     

Occurrence,impact, and elimination of contaminants of emerging concern (CECs) in soil,water, and air streams: advances and challenges in Ibero-American countries

Environmental Science and Pollution Research -     

Maternal-fetal distribution and transfer of dioxins in pregnant women in Japan, and attempts to reduce maternal transfer with Chlorella (Chlorella pyrenoidosa) supplements

Dioxins can be transferred from mother to fetus via the placenta, or to nursing infants via breast milk, potentially causing developmental health problems in children. To assess pediatric health risks from dioxins, exposure of mothers and children to dioxins must be clarified. Methods of reducing maternal transfer of dioxins should also be investigated. Concentrations of 28 dioxin (polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and co-planar polychlorinated biphenyls) congeners in blood, adipose tissue, breast milk, cord blood and placenta collected from 44 pregnant Japanese women were measured. In addition, to investigate potential reductions in maternal transfer of dioxins, 23 pregnant women were instructed to take Chlorella pyrenoidosa supplements during pregnancy. Correlations were observed between dioxin total toxic equivalents (total TEQ) in blood and total TEQ in adipose tissue (r=0.913, P<0.0001), breast milk (r=0.695, P=0.0007), and cord blood (r=0.759, P<0.0001). Dioxin levels transferred to fetuses and nursing infants reflect cumulative maternal concentrations of dioxins. A linear regression equation was introduced to predict total TEQ in breast milk and cord blood from dioxin levels in maternal blood, which should prove useful in evaluating fetal and infant risk of dioxin exposure. Total TEQ in cord blood were approximately 26% lower than in maternal blood (P<0.0001). The results of this study suggest that transplacental transfer differs depending on the dioxin congener. Total TEQ in breast milk were approximately 30% lower in the Chlorella group than in controls (P=0.0113). This finding suggests that maternal transfer of dioxins can be reduced using dietary measures such as Chlorella supplements.     

Effect of alginate on the aggregation kinetics of copper oxide nanoparticles (CuO NPs): bridging interaction and hetero-aggregation induced by Ca<Superscript>2+</Superscript>

The stability of CuO nanoparticles (NPs) is expected to play a key role in the environmental risk assessment of nanotoxicity in aquatic systems. In this study, the effect of alginate (model polysaccharides) on the stability of CuO NPs in various environmentally relevant ionic strength conditions was investigated by using time-resolved dynamic light scattering. Significant aggregation of CuO NPs was observed in the presence of both monovalent and divalent cations. The critical coagulation concentrations (CCC) were 54.5 and 2.9 mM for NaNO₃ and Ca(NO₃)₂, respectively. The presence of alginate slowed nano-CuO aggregation rates over the entire NaNO₃ concentration range due to the combined electrostatic and steric effect. High concentrations of Ca²⁺ (>6 mM) resulted in stronger adsorption of alginate onto CuO NPs; however, enhanced aggregation of CuO NPs occurred simultaneously under the same conditions. Spectroscopic analysis revealed that the bridging interaction of alginate with Ca²⁺ might be an important mechanism for the enhanced aggregation. Furthermore, significant coagulation of the alginate molecules was observed in solutions of high Ca²⁺ concentrations, indicating a hetero-aggregation mechanism between the alginate-covered CuO NPs and the unabsorbed alginate. These results suggested a different aggregation mechanism of NPs might co-exist in aqueous systems enriched with natural organic matter, which should be taken into consideration in future studies.

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Graphical abstract Hetero-aggregation mechanism of CuO nanoparticles and alginate under high concentration of Ca²⁺

     

Azadirachtin induced changes in feeding,dietary utilization and midgut carboxylesterase activity of the final instar larvae of spodoptera litura (fabricius) (lepidoptera: Noctuidae)

Abstract

Final (sixth) instar larvae of Spodoptera litura were fed in increasing amounts of azadirachtin and the effects on feeding activity, dietary utilization and mid‐gut carboxylesterase activity were determined. The antifeedant activity of azadirachtin did not increase in a dose‐dependent manner as reflected by the decline in antifeedant effect at higher doses of 30 and 50 ppm. Dietary concentrations of azadirachtin although significantly lowered the efficiencies of conversion of both ingested (ECI) and digested (ECD) food, it failed to lower the approximate digestibility (AD). The latter however, increased significantly with increasing concentrations of azadirachtin. Feeding of higher doses (30 and 50 ppm) resulted in significant increase in midgut carboxylesterase activity. Thus insensitivity to antifeedant effects and induction of midgut carboxyesterase activity in the older larvae of S.litura In response to high doses of azadirachtin observed in this study can have adverse consequences unless particular attention is paid in developing application strategies for azadirachtin based biopesticides for use in pest control protocols.