Characterization of E1 Kraft Mill Effluent by Toxicity Identification Evaluation Methodology

In order to recover and reuse water in the Kraft mill process, evaluation of separate streams is required to identify toxic compounds or microcontaminants. The stage E1 Kraft effluent, corresponding to the first extraction step of the bleaching Kraft mill process, provides the main toxic compounds found in the final process effluent. This paper uses the toxicity identification evaluation (TIE) procedure for the physicochemical and ecotoxicological characterization of the E1 Kraft effluent. To distinguish the most important toxic compounds, a physicochemical characterization and Phase I of the TIE procedure were performed. The acute toxic effect of the E1 Kraft effluent and treated fraction was performed on Daphnia magna. Results show that untreated E1 Kraft effluent exerts an acute toxic effect on D. magna (24 h LC₅₀?=?27.6%), where the E1 Kraft effluent is characterized by pH 10.5, chemical organic demand (COD) 1,348.8 mg/l, and biological organic demand (BOD₅) 397.5 mg/l, while total phenolic compounds and color are 853.7 mg/l and 0.204 1?×?1 cm, respectively. Additionally, Cu⁺² (0.51 mg/l) and Fe⁺² (0.64 mg/l) were detected. With respect to different treatments, our results indicate that activated carbon, anionic and cationic exchange treatments were able to reduce more that 45% of E1 Kraft effluent’s acute toxicity and that the ethylenediaminetetraacetic acid treatment was able to reduce the E1 Kraft effluent’s acute toxicity to around 75% and the Cu⁺² concentration to 0.019 mg/l. Moreover, specific analysis of heavy metals and organic compounds by GC-MS show that the main compound responsible for the toxicity was Cu⁺², whose tolerance level on D. magna of the 0.12 mg/l.     

Combined effects of technical grade fenitrothion, humic acids and particulate matter on cholinesterase activity in freshwater invertebrates

Purpose

The relative sensitivity of two freshwater invertebrate organisms to the organophosphorus insecticide fenitrothion was assessed by measuring cholinesterase (ChE) activity, a well-known biomarker of both exposure and effect to organophosphorus pesticides. The influence of different concentrations of humic acids (HAs) and particulate matter on fenitrothion bioavailability was assessed in the more sensitive species.

Materials and methods

The selected invertebrates were the dwelling feeding oligochaete Lumbriculus variegatus and the pulmonate gastropod Biomphalaria glabrata. Acute 48-h bioassays were performed exposing organisms to different fenitrothion concentrations. The concentrations that induced 50 % inhibition of enzyme activity (EC₅₀) were calculated. Fenitrothion bioavailability was investigated using different concentrations of commercial HA or particulate matter. Sand and a diverse selection of chromatographic resins that have been proposed as analogues of natural sediments were selected. For these experiments, animals were exposed to a fenitrothion value similar to the EC₅₀.

Results and discussion

The 48-h EC₅₀ values were 12?±?2 and 23?±?3 μg?l^?1 for L. variegatus and B. glabrata, respectively. Depending on HA concentration and the characteristics of particles, ChE activity was similar or higher than the value recorded for animals exposed only to the pesticide in aqueous solution.

Conclusion

The results indicated that L. variegatus was the more sensitive species of the two. In this species, fenitrothion bioavailability did not increase due to the presence of either different HA concentrations or particulate matter. The experimental approach may constitute a useful tool to predict the influence of dissolved organic matter and sediment particles on fenitrothion bioavailability and toxicity to non-target aquatic invertebrates.     

A combined hydraulic and toxicological approach to assess re-suspended sediments during simulated flood events. Part I–multiple biomarkers in rainbow trout

Purpose

One of the central issues related to global changes in weather is the increasing occurrence of flood events that can result in the re-suspension of contaminated sediments in rivers. Here, we report on a proof-of-concept study combining hydraulic engineering and ecotoxicology in a new interdisciplinary approach to assess the toxicity of re-suspended polluted sediments after a simulated flood event.

Materials and methods

Rainbow trout (Oncorhynchus mykiss) were exposed for 5 days under simulated flood conditions in an annular flume with artificial sediments that were spiked with a mixture of polycyclic aromatic hydrocarbons (PAH) at environmentally relevant concentrations. Specifically, the objective of this study was to bridge the gap between the physical re-suspension of pollutants and resulting toxicological impacts on aquatic organisms. A suite of different molecular, biochemical and histological markers was used to test the hypothesis that re-suspension of sediments can lead to re-mobilization of PAHs and subsequently to effects on aquatic organisms.

Results and discussion

The micronucleus frequency was significantly 4.3-fold elevated after exposure. There was no significant indication of Aryl hydrocarbon receptor signaling (no EROD induction or increased CYP1A protein content, only slight induction of CYP1A gene expression). Biliary metabolite concentration was the most sensitive marker of PAH exposure. Results for other biomarkers (glutathione-S-transferase, catalase and lipid peroxidation) were inconclusive.

Conclusions

In combination with chemical analyses of suspended matter, the presented approach will be used to improve understanding of the re-mobilization of pollutants from sediments in support of environmental risk assessment.     

A color removal process for kraft pulp and paper mill effluents

An optimized co-precipitation process for color removal from Kraft pulp and paper mill effluents is described. The process involves sequential addition of alum and lime under controlled conditions, as well as the influence of selected polyelectrolytes on the degree of color removal. Results obtained were in the range of 86 to 95% removal for caustic extract effluent and whole mill effluent. An Al⁺³/Ca⁺² ratio of 0.23 gave optimal results with a final pH in the 4 to 5 range. The chemicals used in the suggested process are commonly available in most Kraft mills. As well, significant reductions in chemical oxygen demand (COD) were observed during the color removal process. An economic analysis indicates that the process is financially attractive.     

Effects of effluents from a sewage treatment plant on the aquatic organisms

The effects of sewage pollution on the distribution and abundance of some orgnanisms over a period of 4 mo at the Ahmadu Bello University Main Campus sewage treatment plant and Kubanni river were studied. Physico-chemical parameters such as dissolved oxygen; temperature; hydrogen ion concentration (pH); electrical conductivity and chemical oxygen demand influenced by pollution were measured with respect to the population of aquatic organisms. The organisms were used as biological indices to monitor the ecological imbalance caused by the effluent. The variations in concentrations of some heavy metals were also measured. The studies revealed that at the source of pollution there were no organisms except for some insect larva such as Eristalis, Psychoda, and some microscopic algae like Oscillatoria, Anaebaena, and Polysystis. A location (site 5) on Kubanni river just before the confluence of the sewage effluent and the river was taken as the control. There was a wide variety of organisms (crustaceans, rotifers, and fish) at the control point.     

Influence of Three Aquatic Macrophytes on Mitigation of Nitrogen Species from Agricultural Runoff

Agricultural runoff containing nitrogen fertilizer is a major contributor to eutrophication in aquatic systems. One method of decreasing amounts of nitrogen entering rivers or lakes is the transport of runoff through vegetated drainage ditches. Vegetated drainage ditches can enhance the mitigation of nutrients from runoff; however, the efficiency of nitrogen removal can vary between plant species. The efficiency of three aquatic macrophytes, cutgrass (Leersia oryzoides), cattail (Typha latifolia), and bur-reed (Sparganium americanum), to mitigate dissolved and total nitrogen from water was investigated. Replicate mesocosms of each plant species were exposed to flowing water enriched with ammonium and nitrate for 6?h, allowed to remain stagnant for 42?h, and then flushed with non-enriched water for an additional 6?h to simulate a second storm event. After termination of the final simulated runoff, all vegetated treatments lowered total nitrogen loads exiting mesocosms by greater than 50%, significantly more than unvegetated controls, which only decreased concentrations by 26.9% (p????0.0023). L. oryzoides and T. latifolia were more efficient at lowering dissolved nitrogen, decreasing ammonium by 42?±?9% and 59?±?4% and nitrate by 67?±?6% and 64?±?7%, respectively. All treatments decreased ammonium and nitrate concentrations within mesocosms by more than 86% after 1?week. However, T. latifolia and L. oryzoides absorbed nitrogen more rapidly, lowering concentrations by greater than 98% within 48?h. By determining the nitrogen mitigation efficiency of different vegetative species, plant communities in agricultural drainage ditches can be managed to significantly increase their remediation potential.     

Heavy Metal Content of Soils and Plum Orchards in an Uncontaminated Area

There are multiple stressors derived from urbanizations that result in frequent disturbances on streams and rivers reducing water quality and threatens aquatic biota. P-glycoprotein (P-gp)-mediated multixenobiotic resistance (MXR) is a defence mechanism analogous to multidrug resistance (MDR), which has been demonstrated in several aquatic organisms. This system protects cells against the entry and the accumulation of xenobiotics and has been proposed as a biomarker for pollution assessment. We conducted a study in a post-urban reach of Esquel stream (Chubut Province) downstream a wastewater treatment plant, in order to assess the presence and activity of MXR in five freshwater macroinvertebrate species (Helobdella michaelseni, Helobdella simplex, Patagoniobdella variabilis, Hyalella curvispina and Chironomus riparius). We measured the accumulation of the model P-gp substrate rhodamine B (RB) in organisms previously exposed to pollution. Our results described the activity of the MXR system in the three species of leeches suggesting their suitability as the in vivo biomonitoring. We also identified a dependence of the transporter activity with the development stage in H. simplex, highlighting the importance of using organisms of similar size classes since it may affect observed results. Finally, we concluded that benthic freshwater macroinvertebrates possess different species-specific levels of MXR activity possibly influencing their natural distribution as well as their survival.     

Assessing the Impact of Effluents from a Modern Wastewater Treatment Plant on Breakdown of Coarse Particulate Organic Matter and Benthic Macroinvertebrates in a Lowland River

Wastewater treatment plants (WWTP) with insufficient technologies for wastewater purification often cause a distinct nutrient pollution in the receiving streams. The increased concentrations of dissolved nutrients can severely disturb the ecological integrity of streams, which has been recently shown for basic ecosystem processes like mineralization of coarse particulate organic matter (CPOM). The present study investigated the impact of a modern WWTP (Zentralkläranlage Jena) on breakdown rates of CPOM exposed in net bags (1 mm mesh size) to the effluent of a large municipal WWTP and an upstream control site in the Saale River (Thuringia, Germany) from April to October 2005. Control and effluent site differed significantly in water chemistry with increased concentrations of dissolved organic carbon (DOC), ammonium, sulfate, and chloride at the effluent site, while the control site displayed higher concentrations of nitrate. However, breakdown rates of toothpickers and small twigs were not significantly different between the sites, whereas breakdown rate of leaf litter was significantly higher at the effluent site (k?=?0.0124 day^?1) than at the control site (k?=?0.0095 day^?1). Benthic invertebrate assemblages inhabiting the sandy stream bed at both sites were dominated by Chironomidae and Oligochaeta, typical inhabitants of fine sediments. Although the Shannon diversity of the benthic invertebrates was slightly higher at the effluent site (0.85) than at the control site (0.63), no significant difference could be detected. Bacterial numbers in water samples and surface biofilms on glass slides also displayed no significant differences between the two sites. This study showed that the effluents of a WWTP with modern technologies for wastewater purification did not directly affect breakdown rates of CPOM, bacteria numbers in epibenthic biofilms and the water column, and the community composition of sediment inhabiting aquatic macroinvertebrates in an effluent-receiving river with already increased concentrations of dissolved nutrients.     

The Morphophysiological Responses of Free-Floating Aquatic Macrophytes to a Supra-optimal Supply of Manganese

Among the many anthropogenic abiotic stresses, manganese (Mn) toxicity has been recognized for its impact on aquatic ecosystems as well as on the biological components of these ecosystems, including aquatic plants. The objective of this study was to determine the Mn accumulation ability of aquatic macrophytes (Azolla caroliniana, Salvinia minima and Spirodela polyrhiza) and evaluate the morphophysiological responses of the species that gather the highest amount of Mn when exposed to a supra-optimal supply of manganese. The experiments were conducted in the laboratory, and the effects of Mn were evaluated based on plant growth; the concentration of total chlorophyll, carotenoids, and anthocyanins; the enzymatic activity of catalase and peroxidase; and leaf anatomy. All of the studied species accumulated Mn in their tissues. Moreover, it was observed that this accumulation was dependent on the concentration of the metal in solution. S. polyrhiza showed higher concentrations of Mn in its tissues (17.062?mg?g^?1 dry weight (DW)), followed by S. minima (4.283?mg?g^?1 DW) and A. caroliniana (1.341?mg?g^?1 DW). Despite the Mn accumulation in all species, S. polyrhiza was the only one selected for further analyses because of its greater ability to accumulate Mn. The high Mn concentration found in tissues of S. polyrhiza suggests that this species has the potential to sequester and accumulate this metal. However, a sensitive response in the plants exposed to higher Mn concentrations (0.4?mM) was observed. The phytotoxicity effects of this accumulation were responsible for a decrease in the plant growth, a reduction in the pigment content (total chlorophyll, carotenoids, and anthocyanins), a low activity of catalase, and the disarrangement of the leaf aerenchyma.     

Comparative Selenium Toxicity to Laboratory-Reared and Field-Collected Hyalella azteca (Amphipoda, Hyalellidae)

Selenium (Se) contamination of aquatic habitats is a global environmental issue. Although organic forms of Se are thought to represent the most bioavailable forms of Se, elevated concentrations of inorganic Se can cause toxicity in aquatic organisms such as benthic invertebrates. To assess the potential role of Se in mortalities observed during previous in situ invertebrate exposures, laboratory experiments on toxicity of inorganic Se (selenate) to Hyalella azteca were performed. Both a laboratory-reared and a field-collected H. azteca population were exposed to Se concentrations ranging from near 0 (control) to 21.79?mg/L, and survival of exposed individuals was monitored over 10?days. In the laboratory-reared H. azteca, significant changes in mortality pattern and reductions in mean survival time (MST) were noted in the test groups exposed to ??0.164?mg/L Se. In the field-collected animals however, significant changes in mortality pattern and reductions in MST were measured in H. azteca exposed to ??1.43?mg/L Se. The 10-day LC50s were 0.086 and 0.574?mg Se/L for the laboratory and field-collected H. azteca populations, respectively. The laboratory-reared group thus was about one order of magnitude more sensitive to Se exposure than the field-collected amphipods. Our results suggest that Se toxicity was likely not a major contributor to amphipod mortalities observed in earlier field studies. Furthermore, population-specific tolerances of the test organisms may need to be considered when extrapolating laboratory-generated data to field situations.     

Ecological chemistry of mercury contained in bleached kraft pulp mill effluents

Chemistry of effluents entering natural water bodies is usually ecological chemistry. The object of its study is the influence of effluents on chemical and biological processes of self-purification. Mercury and its compounds are priority pollutants in bleached kraft pulp mill effluents. It is ascertained that Hg enters the effluents from wood and auxiliary reagents: compounds of Na and active Cl and also from H₂SO₄ employed for neutralization of effluents before biological treatment. At the investigated enterprise the effluents have three stages of treatment: biological, physico-chemical and mechanical. This treatment reduces the Hg concentration in effluents from 10 to 30 ppb to 0.3 to 0.5 ppb. No increased Hg accumulation by aquatic organisms and bottom sediments in the area of effluent discharge was found. Moreover, aquatic organisms grown during 1 mo in the effluents medium did not accumulate Hg in comparison with the control animals. It is found that such ecological effect is obtained as a result of inclusion the modern physico-chemical methods into the complex treatment system.     

Potential for Phosphate Mitigation from Agricultural Runoff by Three Aquatic Macrophytes

Phosphate from agricultural runoff is a major contributor to eutrophication in aquatic systems. Vegetated drainage ditches lining agricultural fields have been investigated for their potential to mitigate runoff, acting similarly to a wetland as they filter contaminants. It is hypothesized that some aquatic macrophytes will be more effective at removing phosphate than others. In a mesocosm study, three aquatic macrophyte species, cutgrass (Leersia oryzoides), cattail (Typha latifolia), and bur-reed (Sparganium americanum), were investigated for their ability to mitigate phosphate from water. Mesocosms were exposed to flowing phosphate-enriched water (10?mg?L^?1) for 6?h, left stagnant for 42?h, and then flushed with non-nutrient enriched water for an additional 6?h to simulate flushing effects of a second storm event. Both L. oryzoides and T. latifolia decreased the load of dissolved phosphate (DP) in outflows by greater than 50?%, significantly more than S. americanum, which only decreased DP by 15?±?6?% (p????0.002). All treatments decreased concentrations inside mesocosms by 90?% or more after 1?week, though the decrease occurred more rapidly in T. latifolia and L. oryzoides mesocosms. By discovering which species are better at mitigating phosphate in agricultural runoff, planning the community composition of vegetation in drainage ditches and constructed wetlands can be improved for optimal remediation results.     

Toxicity of lead nitrate to algae

The fixation of radioactive C was used to mesaure the toxicity of Pb(NO₃)₂ to five species of freshwater algae. Portions of unialgal cultures were inoculated into low salt medium and were used to test all species at 10, 20 and 30 ppm Pb. This medium approximated the salt concentrations of natural aquatic environments. Three different cell weights were used for each concentration of Pb and for the control to determine a relationship between cell weight and toxicity. The concentration of Pb causing a 50% reduction of¹⁴CO₂ fixation as compared to the control was called the ED₅₀ (median effective dose). These values were extrapolated from graphs of ppm Pb vs dpm mg^?1 dry cell weight. The ED₅₀ for three of the species tested (Anabaena, Chlamydomonas andNavicula) was between 15 and 18 ppm Pb. A desmid,Cosmarium, had an ED₅₀ of 5 ppm. This species has a higher surface: volume ratio than the other species tested and this may account for its increased sensitivity. An ED₅₀ forOchromonas was not obtained. Throughout this experiment the fixation of¹⁴CO₂ increased with increasing Pb concentrations and is not readily explained.     

Chronic effects of reduced dissolved oxygen on Daphnia magna

Daphnia magna, initially exposed when less than 48 h old, were maintained at reduced dissolved oxygen (DO) concentrations for 26 days. Exposure was accomplished in a continuous flow recirculating water system. Number of days to first brood, number of young in the first brood, total number of young produced, and dry weight were parameters measured. Statistical analyses show the Daphnia exposed to the lowest DO concentration tested (1.8 mg l^?1) had significantly reduced responses for all parameters measured. In addition, the organisms exposed to 2.7 mg l^?1 O₂ gained less weight than did the controls.     

Ecotoxicological risk assessment of a quarry filling with seaport sediments using laboratory freshwater aquatic microcosms

Purpose

In France, contaminated seaport sediments cannot be discharged into the sea according to recent regulation. Hence, they must be managed on land. Among the solutions identified, that of filling terrestrial quarries located in the littoral zone is one of the most promising. However, this requires developing a methodology for ecological risk assessment; which was the aim of the SEDIGEST research program. In the present study, we report the results of toxicological impacts of three sediments on aquatic ecosystems close to a quarry. These results were used to formulate a risk assessment methodology.

Materials and methods

The ecotoxicological approach was based on laboratory microcosm assays applied to leachates obtained from the sediments. The microcosms contained synthetic water and sediment and five pelagic (micro-algae, duckweeds and daphnids) and benthic (chironomids and amphipods) freshwater species. The biota were exposed for 3 weeks to a range of leachate concentrations; metals were monitored in the water column and the effects (i.e. mortality, growth inhibition and midge emergence) were measured.

Results and discussion

The results showed an absence of acute toxicity at concentrations of <10% (v/v) but sublethal effects for concentrations between 1 and 10%. Given the complex nature of the leachates, it was difficult to determine the factors of toxicity. Metals, especially Cu, might have been responsible for some of the effects on the amphipods.

Conclusions

Future quarries filled with seaport sediments might emit pollutants to aquatic ecosystems in their vicinity, and risk assessors should ensure that predicted environmental concentrations of leachates are below the maximum leachate concentration acceptable for the whole ecosystem; for example, by multiplying the concentration safe for the microcosm organisms by a factor of 10.     

Hydroponic Study of Aluminum Accumulation by Aquatic Plants: Effects of Fluoride and pH

The potential of five common aquatic plant species (Typha latifolia, Myriophyllum exalbescens, Potamogeton epihydrus, Sparganium angustifolium and Sparganium multipedunculatum)to be used for Al phytoremediation was tested. The plants were exposed, for 14 days under hydroponic conditions, to synthetic effluents representing extreme conditions that could occur accidentally at an aluminum refinery site. Tested Al concentrationsranged between 0 and 400 μM, fluoride concentrations between 0 and 900 μM, and the pH varied from 4.5 to 7.5. The results indicate that all the plants tested accumulated aluminum, and as a result induced a decrease of Al in the ambient water. For individual species Al uptake (in mol g^-1 dw d^-1) decreased in the following order: Myriophyllum exalbescens > Sparganium sp. ≌Typha latifolia > Potamogeton epihydrus. M. exalbescens accumulated Al more rapidly in its leaves than in other tissues, whereas T. latifolia and Sparganium sp. accumulated Al essentially in their roots. For P. epihydrus the relative importance of the leaves and roots varied with exposure conditions. For all species, fluoro-Al complexes contributed to Al uptake, contrary to the predictions of the Free-Ion Model, according to which the bioavailability of aluminum should be best predicted by the concentration of the free ion, Al³⁺. The influence of pH on Al uptake varied among the different species and among the parts of the plant: competition between Al and the H⁺-ion was evident for the roots of T. latifolia and the leaves and stem of M. exalbescensand P. epihydrus, whereas the roots of M. exalbescens, P. epihydrus and Sparganium sp. showed an inverse trend (Al uptake increased at low pH). For the leaves of T. latifolia and Sparganium sp., no pH influence could be demonstrated. Overall, the results of this study indicate that aquatic plants have a potential for Al phytoremediation.     

Comparison of arbuscular mycorrhizal and dark septate endophyte fungal associations in soils irrigated with pulp and paper mill effluent and well-water

We compared arbuscular mycorrhizal (AM) and dark septate endophyte (DSE) fungal associations in 2 crops and 31 weeds commonly occurring in pulp and paper mill effluent irrigated and well-water irrigated soils. Soil pH, organic C, N, P and K, were higher in pulp and paper mill effluent irrigated than in well-water irrigated soils. In contrast, the average AM fungal colonization, root length with AM fungal hyphae/hyphal coils, spore numbers and diversity were lower in pulp and paper mill effluent irrigated soils compared to well-water irrigated soils. However, no significant variation was found in DSE fungal colonization nor root length with AM fungal arbuscules/arbusculate coils and vesicles between pulp and paper mill effluent irrigated and well-water irrigated soils. A significant negative correlation existed between AM and DSE fungal colonization in both effluent and well-water irrigated soils. Twelve AM fungal spore morphotypes belonging to Acaulospora, Dentiscutata, Glomus, Racocetra and Scutellospora were isolated from the well-water irrigated soils, whereas spores of six morphotypes were isolated from effluent irrigated soils. AM fungal spore numbers were correlated significantly and positively to AM fungal colonization in effluent and well-water irrigated soils.     

Short-Term Effects of Arsenate-Induced Toxicity on Growth, Chlorophyll and Carotenoid Contents, and Total Content of Phenolic Compounds of Azolla filiculoides

This study evaluated the toxic effects of arsenic (As) on the growth, total antioxidant activity, total content of phenolic compounds, and content of photosynthetic pigments of Azolla filiculoides. The aquatic fern was propagated and exposed to Yoshida nutrient solution contaminated with sodium arsenate (Na₂HAsO₄??7H₂O) at six concentrations (5, 10, 20, 30, 60, and 120???g?As?mL^?1), including the control without As contamination. Azolla cultures were kept under environmental chamber conditions?26??C, 12?h photoperiod and 80% HR for 96?h. Increased As concentrations (>30???g?mL^?1) significantly diminished growth of A. filiculoides and the total content of chlorophyll and total phenolic compounds, but significantly enhanced of total carotenoid?+?xanthophylls content. The concentrations of 5 and 10???g?As?mL^?1 significantly stimulated the growth of A. filiculoides. This aquatic fern tolerates As concentrations lower than 30???g?mL^?1, and its maximum As accumulation (28???g?g^?1 dry weight) was achieved when exposed to 60???g As mL^?1, but showing clear symptoms of As toxicity.     

Stress Protein Responses in South African Freshwater Invertebrates Exposed to Detergent Surfactant Linear Alkylbenzene Sulfonate (LAS)

Stress responses can be measured at various levels of biological organization, from sub-organism through to ecosystem level. This study aimed to investigate stress protein induction as a sub-organism level stress response in two South African freshwater aquatic macroinvertebrates exposed to detergent linear alkylbenzene sulfonate (LAS). Shrimp Caridina nilotica and limpet Burnupia stenochorias were exposed to a range of LAS concentrations (0, 1, 1.8, 3.2, 6.5 and 12.7 mg/L and 0, 0.6, 1.2, 1.6 and 3.2 mg/L respectively) for 96 h. Surviving organisms were prepared for sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis. In C. nilotica there appeared to be induction of a putative ≈ 70 kDa protein at 12.7 mg/L LAS, and induction of putative ≈45 and ≈40 kDa proteins at concentrations of 6.5 mg/L LAS and above. However, only an Hsp70 protein was detected with anti-Hsp72/Hsc73 at 12.7 mg/L LAS. No protein induction was observed in exposed B. stenochorias, however an Hsp40 protein was detected with anti-Hsp40 in exposed and unexposed limpets.