Immunotoxicity of the xenoestrogen 4-nonylphenol to the cockle Cerastoderma glaucum

The in vivo effects of 4-nonylphenol (NP) on functional responses of haemocytes from the cockle Cerastoderma glaucum were investigated after 7 days exposure to sublethal NP concentrations (0, 0+acetone, 0.0125, 0.025, 0.05 and 0.1 mg/l NP). Haemocytes from both controls and exposed cockles were collected, and the effects of NP on total haemocyte count (THC) and volume of circulating cells, intracellular superoxide anion () levels, acid phosphatase and lysozyme-like activities in both haemocyte lysate (HL) and cell-free haemolymph (CFH) were evaluated. Exposure of cockles to 0.1 mg/l NP significantly increased THC (p < 0.05) with respect to controls. Analysis of haemocyte size frequency distribution showed that the haemocyte fraction of about 7–8 μm in diameter and 250 femtolitres in volume increased markedly in cockles exposed to the highest NP concentration tested. Apoptosis resulting in cell volume reduction in NP-exposed animals cannot be excluded. No statistically significant variation in intracellular levels was observed. Conversely, significant increases (p < 0.05) in acid phosphatase activity were observed in CFH from 0.05 and 0.1 mg/l NP-exposed animals; no significant differences in enzyme activity were recorded in HL. Lysozyme-like activity also increased significantly in CFH from cockles exposed to 0.05 mg/l NP (p < 0.05) and 0.1 mg/l NP (p < 0.001). Instead, lysozyme-like activity decreased significantly (p < 0.05) in the HL of animals exposed to 0.05 mg/l NP. Our results suggest that NP induces variations in the functional responses of haemocytes of C. glaucum, mainly by reducing cell membrane stability and promoting cell degranulation.     

Lethal and estrogenic effects of 4-nonylphenol in the cockle Cerastoderma glaucum

The lethal and sublethal effects of the xenoestrogen 4-nonylphenol (NP) were evaluated in the cockle Cerastoderma glaucum. In a 96-h lethality test, bivalves were exposed to 0, 0+ acetone, 0.19, 0.38, 0.75, 1.5 and 3.0 mg NP/l. The 96-h LC(50) value was 0.3mg NP/l. No mortality was observed at 0.1 mg NP/l. The potential estrogenicity of NP was studied in both sexually undifferentiated (resting phase) and differentiated (pre-spawning phase) cockles, exposed for 7 and 14 days to 0, 0+ acetone, 0.0125, 0.025, 0.05, and 0.1 mg NP/l. Vitellogenin (Vg)-like protein levels were determined in both haemolymph and digestive gland by the alkali-labile phosphate (ALP) assay. In the resting phase, exposure for 7 days to 0.1 mg NP/l resulted in significant increases in ALP in both haemolymph and digestive gland, compared with controls. A significant increase was also observed in digestive gland of animals exposed to 0.0125 mg NP/l-exposed animals. After 14 days of exposure, haemolymph ALP levels were significantly increased in exposed animals at all NP concentrations tested, whereas no difference was recorded in digestive gland. In the pre-spawning phase, exposure for 7 days to NP significantly increased ALP levels in haemolymph from males exposed at all NP concentrations tested, whereas no significant variations were found in haemolymph from females. NP (0.05 and 0.1 mg/l) was also shown to increase ALP concentrations significantly in digestive gland of males, but not in those of females. Likewise, after 14 days' exposure, ALP levels significantly increased in haemolymph from males only at 0.1 mg NP/l. Conversely, NP caused significant increases in ALP levels in digestive gland from both males (at all NP concentrations tested) and females (at 0.025 and 0.1 mg NP/l). These results demonstrate that NP induces Vg synthesis in C. glaucum. Interestingly, males were more responsive to NP than females.     

Lethal and estrogenic effects of 4-nonylphenol in the cockle Cerastoderma glaucum

The lethal and sublethal effects of the xenoestrogen 4-nonylphenol (NP) were evaluated in the cockle Cerastoderma glaucum. In a 96-h lethality test, bivalves were exposed to 0, 0+ acetone, 0.19, 0.38, 0.75, 1.5 and 3.0 mg NP/l. The 96-h LC₅₀ value was 0.3 mg NP/l. No mortality was observed at 0.1 mg NP/l. The potential estrogenicity of NP was studied in both sexually undifferentiated (resting phase) and differentiated (pre-spawning phase) cockles, exposed for 7 and 14 days to 0, 0+ acetone, 0.0125, 0.025, 0.05, and 0.1 mg NP/l. Vitellogenin (Vg)-like protein levels were determined in both haemolymph and digestive gland by the alkali-labile phosphate (ALP) assay. In the resting phase, exposure for 7 days to 0.1 mg NP/l resulted in significant increases in ALP in both haemolymph and digestive gland, compared with controls. A significant increase was also observed in digestive gland of animals exposed to 0.0125 mg NP/l-exposed animals. After 14 days of exposure, haemolymph ALP levels were significantly increased in exposed animals at all NP concentrations tested, whereas no difference was recorded in digestive gland. In the pre-spawning phase, exposure for 7 days to NP significantly increased ALP levels in haemolymph from males exposed at all NP concentrations tested, whereas no significant variations were found in haemolymph from females. NP (0.05 and 0.1 mg/l) was also shown to increase ALP concentrations significantly in digestive gland of males, but not in those of females. Likewise, after 14 days’ exposure, ALP levels significantly increased in haemolymph from males only at 0.1 mg NP/l. Conversely, NP caused significant increases in ALP levels in digestive gland from both males (at all NP concentrations tested) and females (at 0.025 and 0.1 mg NP/l). These results demonstrate that NP induces Vg synthesis in C. glaucum. Interestingly, males were more responsive to NP than females.     

In vitro effects of nonylphenol on functional responses of haemocytes of the colonial ascidian Botryllus schlosseri

The effects of nonylphenol (NP) on functional responses of haemocytes of the colonial ascidian Botryllus schlosseri were evaluated. Haemocytes were exposed to 0.1, 1 and 10 μM NP and the effects on haemocyte viability, adhesion, morphology, lysosomal membrane stability, phagocytic activity and early apoptosis were evaluated. Haemocyte viability and adhesion were not affected by NP. Phagocytic activity and the amoebocytic index decreased significantly at all the concentrations tested, while exposure of haemocytes to 1 and 10 μM NP caused a significant increase in the diffusion of Neutral Red into the cytosol. The percentage of cells positive to Annexin-V (indicative of early apoptosis) increased significantly at 1 and 10 μM NP. Results obtained suggested a relationship between NP and alterations in functional responses of haemocytes in B. schlosseri. Biomarkers measured resulted sensitive, rapid and reproducible, even if their responsiveness will be evaluated after in vivo exposure of animals to NP.     

Lethal and estrogenic effects of 4-nonylphenol in the cockle Cerastoderma glaucum

The lethal and sublethal effects of the xenoestrogen 4-nonylphenol (NP) were evaluated in the cockle Cerastoderma glaucum. In a 96-h lethality test, bivalves were exposed to 0, 0+ acetone, 0.19, 0.38, 0.75, 1.5 and 3.0 mg NP/l. The 96-h LC₅₀ value was 0.3 mg NP/l. No mortality was observed at 0.1 mg NP/l. The potential estrogenicity of NP was studied in both sexually undifferentiated (resting phase) and differentiated (pre-spawning phase) cockles, exposed for 7 and 14 days to 0, 0+ acetone, 0.0125, 0.025, 0.05, and 0.1 mg NP/l. Vitellogenin (Vg)-like protein levels were determined in both haemolymph and digestive gland by the alkali-labile phosphate (ALP) assay. In the resting phase, exposure for 7 days to 0.1 mg NP/l resulted in significant increases in ALP in both haemolymph and digestive gland, compared with controls. A significant increase was also observed in digestive gland of animals exposed to 0.0125 mg NP/l-exposed animals. After 14 days of exposure, haemolymph ALP levels were significantly increased in exposed animals at all NP concentrations tested, whereas no difference was recorded in digestive gland. In the pre-spawning phase, exposure for 7 days to NP significantly increased ALP levels in haemolymph from males exposed at all NP concentrations tested, whereas no significant variations were found in haemolymph from females. NP (0.05 and 0.1 mg/l) was also shown to increase ALP concentrations significantly in digestive gland of males, but not in those of females. Likewise, after 14 days’ exposure, ALP levels significantly increased in haemolymph from males only at 0.1 mg NP/l. Conversely, NP caused significant increases in ALP levels in digestive gland from both males (at all NP concentrations tested) and females (at 0.025 and 0.1 mg NP/l). These results demonstrate that NP induces Vg synthesis in C. glaucum. Interestingly, males were more responsive to NP than females.     

Immune responses to combined effect of hypoxia and high temperature in the green-lipped mussel Perna viridis

Flow cytometry was used to examine immune responses in haemocytes of the green-lipped mussel Perna viridis under six combinations of oxygen level (1.5 mg O2 l(-1), 6.0 mg O2 l(-1)) and temperature (20 °C, 25 °C and 30 °C) at 24 h, 48 h, 96 h and 168 h. The mussels were then transferred to normoxic condition (6.0 mg O2 l(-1)) at 20 °C for further 24 h to study their recovery from the combined hypoxic and temperature stress. Esterase (Est), reactive oxygen species (ROS), lysosome content (Lyso) and phagocytosis (Pha) were reduced at high temperatures, whereas hypoxia resulted in higher haemocyte mortality (HM) and reduced phagocytosis. For HM and Pha, changes were observed after being exposed to the stresses for 96 h, whereas only a 24 h period was required for ROS and Lyso, and a 48 h one for Est. Recovery from the stresses was observed for HM and Pha but not other immune responses.     

Effects of 4-nonylphenol exposure in mussels (Mytilus galloprovincialis) and crabs (Carcinus aestuarii) with particular emphasis on vitellogenin induction

Since it is often difficult to estimate possible adverse effects due to contamination in selected ecosystems, multi-species biomonitoring may provide more information, taking into account different routes of exposure, ecological roles and metabolic capabilities of animals. In this context, we exposed for 7 days the mussel Mytilus galloprovincialis and the crab Carcinus aestuarii to 4-nonylphenol (NP), a well-known xenoestrogen. In mussels (0-0.2 mg NP l(-1)), we measured NP bioaccumulation in soft tissues and vitellogenin (Vg)-like protein levels in digestive glands from both males and females by the alkali-labile phosphate assay (ALP). As no reference data were available for crab exposure, the NP 96-h LC(50) value was previously determined. Then, in sublethally exposed (0-1.0 mg NP l(-1)) male crabs, NP bioaccumulation and Vg levels were measured in hemolymph, gonads and digestive gland. Bioaccumulation of NP increased from 43 to 371 microg g(-1) d.w. in mussels, and from 3.6 to 37 microg g(-1) d.w. in crabs, depending on the NP concentration in water. Dose-dependent Vg-like protein induction was observed in both species, appearing to be related to NP bioaccumulation, although a partial decrease was recorded at the highest concentration tested. A similar trend was observed in both digestive gland and gonad of exposed crabs; Vg increased to a lesser extent, although significantly, in hemolymph. Results demonstrated that NP induces Vg synthesis both in male and female mussels, as well as in male crabs. On the basis of the responsiveness of both species investigated, a multi-species approach is indicated in biomonitoring programmes.     

Sublethal impact of short term exposure to the organophosphate pesticide azamethiphos in the marine mollusc Mytilus edulis

Concern has been raised that the increased use of pesticides in intensive aquaculture practices may cause adverse sublethal effects to non-target aquatic species. Azamethiphos is an organophosphate (OP) pesticide used to combat sea lice infestations in farmed salmonids. Here, the sublethal impact on the blue mussel, Mytilus edulis, of short term exposure to azamethiphos was determined. The testing regime included biomarkers of exposure (acetylcholinesterase activity), cytotoxicity (neutral red retention), immune function (phagocytic index) and physiological condition (feeding rate). The distribution and sensitivity of M. edulis acetylcholinesterase to inhibition by azamethiphos was first determined, yielding IC(50) values of 0.736 and 1.30 mg l(-1) for gill and haemolymph, respectively. Exposure of mussels to 0.1 mg l(-1) azamethiphos for periods of up to 24h caused a significant reduction in acetylcholinesterase activity in both the haemolymph (P<0.0002) and the gill (P<0.002), alteration in cell viability (P<0.02) and decrease in phagocytic index (P<0.03). The feeding rate remained unaffected. The results support the hypothesis that, in addition to its neurotoxic effects, azamethiphos can modulate haemocyte function and immune defence in M. edulis at environmentally relevant concentrations after only a few hours.     

Inhibition of Fertilization and Larval Metamorphosis of the Coral Acropora millepora (Ehrenberg, 1834) by Petroleum Products

Accidental oil spills from ships or rigs and inputs of effluent such as production formation water (PFW) are key perceived threats to tropical biota from industry activities. Scleractinian corals are an important functional component of tropical reefs and the abundance, diversity and resilience of coral communities can be used as an indicator of ecosystem health. In this paper, we report the effects of petroleum products, including water accommodated fractions (WAF) of crude oil, PFW and dispersant (Corexit 9527), on fertilization and larval metamorphosis of the widespread scleractinian coral, Acropora millepora (Ehrenberg, 1834) in laboratory-based assays. At 20% v/v PFW fertilization was inhibited by 25%. This concentration was equivalent 0.0721 mg l⁻¹ total hydrocarbon (THC). In contrast, larval metamorphosis was more sensitive to this effluent, with 98% metamorphosis inhibited at the same concentration. Crude oil WAF did not inhibit fertilization of gametes until dispersant was introduced. Dispersed oil was slightly more toxic to fertilization than dispersant alone, suggesting toxicity to that event may be additive. The minimum concentration of dispersed oil which inhibited fertilization was 0.0325 mg l⁻¹ THC. Larval metamorphosis was more sensitive than fertilization to crude oil. Although crude oil and dispersant inhibited larval metamorphosis individually, this toxicity was magnified when larvae were exposed to combinations of both. Crude oil inhibited metamorphosis at 0.0824 mg l⁻¹ THC and at 0.0325 mg l⁻¹ THC when dispersed in 10% v/v (dispersant/oil). Management of petroleum-related risks to spawning corals should consider not only the occurrence of the annual coral spawning event, but also the subsequent 1–3-week period during which most larval metamorphosis and recruitment occur.     

Exposure of the clam Tapes philippinarum to 4-nonylphenol: changes in anti-oxidant enzyme activities and re-burrowing capability

The effects of 4-nonylphenol on anti-oxidant enzyme (superoxide dismutase and catalase) activity in both gills and digestive gland and re-burrowing capability of the clam Tapes philippinarum were investigated after 7 days' exposure to various sublethal concentrations of nonylphenol (NP). NP caused a significant decrease in superoxide dismutase activity in both tissues from the lowest concentration tested, whereas no significant alteration with respect to controls was observed in catalase activity. NP effects on re-burrowing of clams were evaluated in two experiments. In the first, clams exposed to the highest NP concentrations showed a marked delay in re-burrowing 48 h after the end of exposure. Modifications in normal burrowing behaviour were also observed, suggesting that NP acts via narcosis and/or neurotoxic mechanisms. In the second experiment, the elapsed time for 50% of the exposed clams to re-burrow into the sediment (RT(50)) was also calculated in a 24-h re-burrowing response test. At the highest NP concentrations, it was not possible to estimate RT(50) values, as the percentage of clams showing complete burial was less than 50%. The present study demonstrates that NP causes oxidative stress by inhibiting superoxide dismutase activity and greatly affects the re-burrowing capability of T. philippinarum, even at environmentally realistic concentrations.     

Estrogen equivalent concentration of individual isomer-specific 4-nonylphenol in Ariake sea water, Japan

Concentrations of 4-nonylphenol (NP) were determined by isomer-specific quantification of individual NP isomers based on relative response factor (RRF) quantification with GC–MS in combination with steam distillation extraction. Concentrations of NP in the Ariake Sea decreased with distance from the river mouth (St.A; 49 ng NP/l) to offshore areas (St.C; 11 ng NP/l). Even the least concentration in water from St.C in Ariake Sea was sufficient to have adverse effects on barnacles. The isomers, NP1–NP14 were separated by GC–PFC and identified structurally with NMR. The isomers varied in estrogenic activity with NP7 exhibiting the greatest estrogenic activity with a potency that was approximately 1.9 × 10⁻³ that of 17β-estradiol (E2) in recombinant yeast screen system. The coefficient of variation (CV) of NP isomer’s concentrations among three samples at St.A, B and C were 4–75%. This suggests that NP isomers might be independently degraded in aquatic environmental samples. The predicted estrogenic activity of measured concentrations of NP in Ariake Sea was 2.7–3.0-fold greater than the measured estrogen agonist activity.     

Effects of 4-nonylphenol exposure in mussels (Mytilus galloprovincialis) and crabs (Carcinus aestuarii) with particular emphasis on vitellogenin induction

Since it is often difficult to estimate possible adverse effects due to contamination in selected ecosystems, multi-species biomonitoring may provide more information, taking into account different routes of exposure, ecological roles and metabolic capabilities of animals. In this context, we exposed for 7 days the mussel Mytilus galloprovincialis and the crab Carcinus aestuarii to 4-nonylphenol (NP), a well-known xenoestrogen. In mussels (0–0.2 mg NP l⁻¹), we measured NP bioaccumulation in soft tissues and vitellogenin (Vg)-like protein levels in digestive glands from both males and females by the alkali-labile phosphate assay (ALP). As no reference data were available for crab exposure, the NP 96-h LC₅₀ value was previously determined. Then, in sublethally exposed (0–1.0 mg NP l⁻¹) male crabs, NP bioaccumulation and Vg levels were measured in hemolymph, gonads and digestive gland. Bioaccumulation of NP increased from 43 to 371 μg g⁻¹ d.w. in mussels, and from 3.6 to 37 μg g⁻¹ d.w. in crabs, depending on the NP concentration in water. Dose-dependent Vg-like protein induction was observed in both species, appearing to be related to NP bioaccumulation, although a partial decrease was recorded at the highest concentration tested. A similar trend was observed in both digestive gland and gonad of exposed crabs; Vg increased to a lesser extent, although significantly, in hemolymph. Results demonstrated that NP induces Vg synthesis both in male and female mussels, as well as in male crabs. On the basis of the responsiveness of both species investigated, a multi-species approach is indicated in biomonitoring programmes.     

Protective effect of phospholipid hydroperoxide glutathione peroxidase (PHGPx) against lipid peroxidation in mussels Perna perna exposed to different metals

Levels of antioxidant defenses and lipid peroxidation were evaluated in mussels exposed to lead (200 mg/l), iron (500 microg/l), cadmium (200 microg/l) and copper (40 microg/l), for 12, 24, 72 and 120 h. Glutathione S-transferase (GST) activity was unchanged with all treatments. Catalase (CAT) increased after 120 h of exposure to all metals. Mussels exposed to Cd for 12 h, and to Cu and Fe for 120 h had increased lipid peroxidation, which might be associated to decreased levels of reduced glutathione (GSH) and glutathione peroxidase (GPx) activity. Pb exposure caused GSH depletion after 12 h and increased GPx activity after 120 h. Negative correlations were observed between the enzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) and malonaldehyde (MDA) levels after Fe and Cu exposure, indicating a protective role of PHGPx against lipid peroxidation, and suggesting the use of this enzyme as a new potential biomarker of toxicity associated with contaminant exposure in mussels.     

“Man and Biosphere” — Studies on Sikkim Himalayas. Part 2: Acute Toxity of Mixed Copper-Zinc Solutions on Common Carp,Cyprinus carpio (LINN.)

In batch experiments for 6 to 48 h, individuals of Cyprinus carpio (2 g individual weight) were exposed to mixed solutions of copper and zinc sulphate: pH = 6.3, temperature 15 °C, 6.8 mg/l O₂, 7.1 mg/l Ca²⁺, 0.7 mg/l Mg²⁺. The LC₅₀ for the individual substances on exposures of 24 and 48 h amount to 9.04 and 7.28 mg/l, resp., of Zn and 0.11 and 0.095 mg/l, resp., of Cu. The sum of the biological activity of the two kinds of metal ions is assessed according to MARKING'S index: at low concentrations of the mixed solutions the two metals show an additive effect, at higher concentrations a synergistic effect occurs.     

Towards a more ecologically relevant assessment of the impact of heavy metals on the photosynthesis of the seagrass,Zostera capricorni

This in situ study used photosynthetic activity (measured as chlorophyll a fluorescence) and photosynthetic pigment concentrations to assess the effect of copper, cadmium, lead and zinc on the seagrass Zostera capricorni. Custom-made portable in situ exposure (PIE) chambers were developed so seagrasses could be dosed within the meadow. Z. capricorni was exposed to 0.1 and 1 mg l⁻¹ of metal solutions for 10 h. During this time and for the subsequent four-day recovery period, the effective quantum yield of photosystem II (PS II) (ΔF/Fm^′) was measured. While the results were variable, copper and zinc exposed samples had a depressed ΔF/Fm^′ during the exposure period. Samples exposed to zinc recovered to pre-exposure levels but those exposed to copper did not. Cadmium and lead did not impact on the chlorophyll a fluorescence and the chlorophyll pigment data supported these findings. This study presents an innovative new application of chlorophyll a fluorescence stress assessment.     

Changes in the scope for growth of blood cockles (Anadara granosa) exposed to industrial discharge

Blood cockles (Anadara granosa) were placed in plastic cages and deployed at eight locations along a pollution gradient. A control site was chosen a few kilometres away. At 2-week intervals for 8 weeks, the scope for growth of the cockles was determined. A total of 11 environmental parameters were measured during each sampling exercise for correlation with the growth index. Values of scope for growth for all the eight stations, except for stations 7 and 8, were significantly lower than the value for the control site; station 1 (nearest the discharge point) had the lowest value. Strong correlations were found between the scope for growth of the cockles and seven of the environmental parameters. The pattern of the impact of the discharge on the scope for growth of the cockles suggests that longshore currents may play a dominant role in the distribution of the industrial wastes.     

食物浓度对模糊秀体溞生长和繁殖的影响

模糊秀体潘是热带水体中枝角类的主要优势种,它在竞争及对热带水体的适应方面的优势尚不清楚。本文以模糊秀体潘为研究对象,观察不同食物浓度下其生长和繁殖的特点,探讨模糊秀体潘成热带水体优势种的原因。以蛋白核小球藻为食物,浓度梯度设为0.05mg／L、0.1mg／L、0.3mg／L、0.6mg／L和1.0mg／L小球藻浓度为0.05mg／L的培养液中,模糊秀体潘存活率很低且未观察到繁殖现象其它4个浓度小球藻喂养的模糊秀体潘体长生长率和最大体长均随食物浓度的升高而增加,最大体长分别达到1.087mm、1.125mm、1.225mm、1.313mm;食物浓度对模糊秀体溢的胚胎发育时间,平均寿命,个体累计产幼数无显著影响：随着食物浓度的升高,模糊秀体滏初次怀卵体长显著增加,繁殖前期的历时相应缩短,平均每胎产幼数增加：内禀增长率随食物浓度升高而增大,达到0.6mg／L后比较稳定。浓度为0.6mg／L时,模糊秀体潘的净生殖率也达到最大值。1.0mg／L组的世代时间最短,内禀增长率接近最大值。0.6mg／L和1.0mg／L为模糊秀体潘生长比较适宜的食物浓度。热带自然水体中,模糊秀体潘相对其它浮游动物个体小和怀卵率低的现象并非食物不足所致;食物充足条件下的最大成体的个体大小与野外样品接近,也说明自然水体中捕食并没有显著影响成体的个体大小。模糊秀体潘体型较小、身体透明、行动敏捷,容易逃避鱼类的捕食,可能是模糊秀体潘能在食物丰富的热带水体中,特别是透明度较低的富营养化湖泊中能够成为枝角类中优势种的主要原因。     

The weathering rates of some sandstone cliffs central Weald,England

Short-term (1-30 months) weathering rates were obtained for steep cliffs of Cretaceous Ardingly Sandstone in southern England, using a new photographic method. Mean rates for an exposed and a sheltered outcrop were 0.11 and 0.033 mm a^?1 respectively. Significant (p < 0.1) positive correlations were obtained with illumination and algal biomass and a significant negative correlation (p < 0.05) with slope angle. There was no correlation with grain size, porosity, or mean surface temperature. Highest weathering rates were observed on south- and west-facing exposed cliffs. The results are discussed with reference to algal growth, climate, and wind direction.     

Histopathological Changes Induced by Pentachlorophenol and Sodium Pentachlorophenate in the Mantle of the Freshwater Snail Viviparus bengalensis (L.)

The freshwater pondsnail Viviparus bengalensis (LINNAEUS) was exposed to sublethal concentrations of pentachlorophenol (1.561 mg/l) and sodium pentachlorophenate (0.991 mg/l). Both the chemicals caused considerable damage in the mantle of the snail. Their toxic effects included shrinkage and elongation of epithelial cells, the cells losing contact with each other, resulting in the formation of intercellular spaces, enlargement of nuclei, shrinkage of the basement layer and loss of shape of polygonal cells of connective tissue.