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.     

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.     

Responses of the green-lipped mussel Perna viridis (L.) to suspended solids

Laboratory experiments were conducted to investigate the lethal and sublethal effects of suspended solids on the survival and physiological, behavourial and morphological changes of the green-lipped mussel Perna viridis collected from Tolo Harbour, Hong Kong. Results showed that P. viridis survived in all test conditions of suspended solids from 0 to 1200 mg/l over a period of 96 h. Physiological responses of the green-lipped mussel under 14-d exposure of suspended solids from 0 to 600 mg/l, followed by 14-d recovery in natural seawater, revealed no significant changes (p>0.05) in oxygen consumption and dry gonosomatic index for treatments in different concentrations of suspended solids and exposure time. Changes in clearance rate were only found to be significant (p<0.001) with exposure time. Responses in behavourial and morphological changes of the green-lipped mussel were also studied under similar experimental treatments and exposure time. Byssus production was significantly (p<0.001) related to exposure time. Gill damage, however, was significantly greater in treatments (p<0.001). Present findings suggested that P. viridis could tolerate a high level of suspended solids in the laboratory. There were dose-dependent effects of suspended solids on morphology of gill filaments. Implications of survival and responses of the green-lipped mussel to suspended solids in the marine environment are discussed.     

Acute toxicity of cadmium to Channa punctatus (BLOCH)

Effects of acute cadmium poisoning on survival, its residual values and histopathology in certain organs of a freshwater airbreathing fish, Channa punctatus (BLOCH ) were investigated. The threshold concentration, MATC and LC₅₀ values obtained from 96 h static bioassay, revealed that Channa is more susceptible to cadmium ions at higher temperature. The atomic absorption spectrophotometric determination of cadmium residues differed significantly in organs of specimens having survived and died after 96 h of exposure. The gill accumulate the highest amount of cadmium, the liver accumulated a slightly smaller amount than the gill, while the kidney accumulated the least. The histopathological lesions subjected to sublethal (5.2 mg/l Cd) and lethal (8.4 mg/l Cd) concentrations of cadmium were: detachment and rupture of lamellar epithelium, collapse of pillar cells and hypertrophy in mucus producing gland cells in the gill; vacuolization and coagulative necrosis in hepatic cells of the liver; and expansion, necrosis and accumulation of cellular debris in renal tubules of the kidney. The probable causes for death of fish due to cadmium ions have been discussed.     

Alterations in the architecture of the gill surface produced by water-borne copper in Heteropneustes fossilis (BLOCH)

In the present investigation the effect of water-borne copper on the gill architecture of a freshwater teleost, Heteropneustes fossilis (BLOCH ) was studied through scanning electron microscopy. The changes produced on the gill surface in a sublethal measured copper concentration (8.17 mg/1) in static bioassay after 96 h of exposure were fusion of secondary lamellae, rupture of various cellular components and deposition of excessive mucus and blood on the gill surface. The epithelial surface of treated gill thus lost the organized structures like microvilli, arborizing ridges and channels. The cause of fish mortality due to action of copper has been discussed in reference to reduced relative diffusing capacity along with other wide ranging effects on the metabolism of fish.     

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 (O(2)(-)) levels, acid phosphatase and lysozyme-like activities in both haemocyte lysate (HL) and cell-free haemolymph (CFH) were evaluated. Exposure of cockles to 0.1mg/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 microm 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 O(2)(-) levels was observed. Conversely, significant increases (p<0.05) in acid phosphatase activity were observed in CFH from 0.05 and 0.1mg/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/lNP. 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.     

Integrated biomarker assessment of the effects exerted by treated produced water from an onshore natural gas processing plant in the North Sea on the mussel Mytilus edulis

The biological impact of a treated produced water (PW) was investigated under controlled laboratory conditions in the blue mussel, Mytilus edulis. Mussel health status was assessed using an integrated biomarker approach in combination with chemical analysis of both water (with SPMDs), and mussel tissues. Acyl-CoA oxidase activity, neutral lipid accumulation, catalase activity, micronuclei formation, lysosomal membrane stability in digestive cells and haemocytes, cell-type composition in digestive gland epithelium, and the integrity of the digestive gland tissue were measured after 5 week exposure to 0%, 0.01%, 0.1%, 0.5% and 1% PW. The suite of biomarkers employed were sensitive to treated PW exposure with significant sublethal responses found at 0.01-0.5% PW, even though individual chemical compounds of PW were at extremely low concentrations in both water and mussel tissues. The study highlights the benefits of an integrated biomarker approach for determining the potential effects of exposure to complex mixtures at low concentrations. Biomarkers were integrated in the Integrative Biological Response (IBR/n) index.     

An evaluation of hemolymph cholinesterase activities in the tropical scallop,Euvola (Pecten) ziczac,for the rapid assessment of pesticide exposure

The use of sequential measurements of hemolymph cholinesterase activities as a non-invasive biomarker of seasonal organophosphate/carbamate exposure was investigated for the tropical scallop, Euvola (Pecten) ziczac. Overall activities of both acetylcholinesterase and butyrylcholinesterase were relatively high compared to studies with bivalve tissues. Acute in vivo experiments showed inhibition of hemolymph acetylcholinesterase activity at concentrations of the organophosphate insecticide chlorpyrifos of 0.1, 1 and 10 ng l(-1). Monthly sampling of hemolymph from scallops at two sites in Bermuda over a 15 month period showed seasonal acetylcholinesterase and butyrylcholinesterase inhibition. Direct and indirect evidence suggests that this inhibition did not relate to biochemical or physiological changes associated with gonad maturation and spawning, but rather reflected diffuse contamination of the marine environment by cholinesterase inhibitors or increased bioavailability of such inhibitors at these times. Repetitive sampling of scallop hemolymph for cholinesterase activities represents a rapid, sensitive and non-invasive method for assessing seasonal, sublethal pesticide exposure in these commercially important bivalves and suggests a wider use in marine pollution monitoring.     

The effect of petroleum hydrocarbons on reproduction of an estuarine planktonic copepod in laboratory cultures

The effect of short-term exposure to high concentration (mg l.^?1) of water soluble fraction of aromatic heating oil on subsequent egg production by the estuarine copepod Eurytemora affinis has been studied in laboratory cultures to investigate possibly sublethal biological effects following exposure to hydrocarbon under an oil spill. Significant reduction in subsequent length of life, total number of eggs produced, mean brood size, and rate of egg production was observed. Exposure to naphthalene alone at 1 mg l.^?1 for 24 h produced a significant effect on total fecundity of the females. Ingestion rates were significantly lower when measured in the presence of the water soluble fraction, and also naphthalene alone at mg l.^?1 concentrations. Exposure to low-levels (10 and 50 μg l.^?1) of ¹⁴C-naphthalene alone over a period of ten days produced no significant effect on feeding or reproduction despite the high concentrations of hydrocarbon accumulated during this period. The results are discussed in relation to previous work on the fate of hydrocarbons in planktonic copepods, using ¹⁴C-1-naphthalene as a model compound.     

Sublethal effects of cupric ion activity on the grazing behaviour of three calanoid copepods

This research (1) characterized the effects of sublethal cupric ion activities on the grazing behavior of two estuarine copepods (Acartia tonsa, Acartia hudsonica) and one nearshore, neritic copepod (Temora longicornis) and (2) compared the sensitivity of short-term sublethal behavioral assays with that of longer-term acute toxicity tests. A nitrilotriacetate-trace-metal-ion buffer system at 27‰. S was used to quantify and control the free cupric ion activity. Acute toxicity tests were used to determine the mortality of A. tonsa and T. longicornis over 72 h within the approximate cupric ion activity range of 10⁻¹³ to 10^−9.5 M. 24 h survival was not affected within the approximate cupric ion activity range of 10⁻¹³ to 10^−9.7 M, the range used for subsequent grazing activity experiments after 24 h exposure to Cu. Grazing activity was significantly diminished at cupric ion activities of ≈ 10⁻¹⁰ M for A. tonsa and T. longicornis, and at ≈ 10⁻¹¹ M for A. hudsonica. A hormetic pattern of response in feeding activity was observed with A. tonsa and T. longicornis. Grazing activity was found to be a sensitive measure of sublethal Cu stress compared with the acute toxicity tests. Grazing activity was affected at environmentally relevant cupric ion activities.     

Deleterious effects of Malathion on survivality and growth of the fingerlings of Channa punctatus (BLOCH.), a freshwater murrel

The LC₅₀ of Malathion for Channu punctatus is determined for 24… 96 h of exposure as 10.95… 3.22 mg/l. At exposure to 2.5 mg./l Malathion, growth–measured by increase in weight–is significantly reduced from the 19th day and reduced by 10 % till the 31st day. During the same period of time, the RNA and protein contents of the muscle is lowered by 10 and 15 %, respectively, as compared with the control. In addition to an acute toxic effect, in sublethal concentrations Malathion has also pronounced effects on growth and metabolism.     

Field validation, in Scotland and Iceland, of the artificial mussel for monitoring trace metals in temperate seas

The artificial mussel (AM), a novel chemical sampling device, has been developed for monitoring dissolved trace metals in marine environments. The AM consists of Chelex-100 suspended in artificial seawater within Perspex tubing and enclosed with semi-permeable polyacrylamide gel at both ends. To validate the field performance of the AM in temperate waters, we deployed AMs alongside transplanted blue mussels Mytilus edulis in coastal environments in Scotland (Holy Loch, Loch Fyne, Loch Striven and Millport) and Iceland (Reykjavikurh?fn, Gufunes, South of thornerney, Hofsvik, Hvalfj?rethur and Sandgerethi) for monitoring trace metals. While uptake patterns of Cd between the AM and M. edulis were highly comparable, discrepancies were found in the accumulation profiles of the other metals (Cu, Cr, Pb and Zn), in particular Zn. Nonetheless, the AMs gave a better resolution to accurately reveal the spatial difference in dissolved metal contamination when compared with M. edulis. AMs complement the use of mussels since AMs indicate dissolved metals in seawater, whereas uptake by mussels indicates a mixture of dissolved and particulate metals. Our results also indicated that historical metal exposure of the transplanted M. edulis could significantly confound their metal concentrations especially when the deployment period was short (i.e. <34d). This study suggested that the AM can overcome problems associated with variable biological attributes and pre-exposure history in the mussel, and provides a standardized and representative time-integrated estimate of dissolved metal concentrations in different marine environments.     

Acute and chronic toxic effects of the carbamate pesticide Sevin on some haematological,biochemical and enzymatic parameters in the fresh water teleost fish Channa punctatus

The effects of exposure to LC₅₀ (10.5 mg/l) for 96 h and to a sublethal concentration (1.05 mg/l) of Sevin for 120 days have been observed on some haematological, biochemical and enzymatic parameters of Channa punctatus. The fish were hyperglycemic and hyperlactemic and the glycogen content of the liver and muscles decreased, but the lactic acid content of two tissues increased. The activity of hexokinase increased in the kidney, intestine, liver and muscles but decreased in the gills and brain. The activity of lactate dehydrogenase decreased in the kidney and intestine, and increased in the liver, muscles, brain and gills. The pyruvate dehydrogenase activity decreased in all the six tissues. Both the exposures produced a decrease in the succinate dehydrogenase activity of the liver. The enzyme activity decreased in the brain, gills and muscles after chronic exposure, whereas the kidney and intestine showed an elevation in enzyme activity.     

First evidence of altered vitellogenin-like protein levels in clam Tapes philippinarum and in cockle Cerastoderma glaucum from the Lagoon of Venice

Possible xenoestrogenic effects were investigated, for the first time, in two bivalve species from the Lagoon of Venice (Italy): the clam Tapes philippinarum and the cockle Cerastoderma glaucum. Bivalves were collected far from their reproductive phase at the very early stage of gametogenesis (January), and in the pre-spawning period (June) in six sites. Vitellogenin (Vg)-like proteins (a biomarker of exposure to estrogenic compounds) were measured by the alkali-labile phosphate method (ALP), in both haemolymph and digestive gland from males and females (when it was possible to distinguish sex by microscopic observation of gonadal tissue). Haemolymph calcium concentrations (a parameter considered closely related to the presence of Vg-like proteins) and the bivalve condition index (CI) were also measured. In both seasons, bivalves collected at Campalto (near a sewage treatment plant) and Marghera (a highly contaminated area) had higher Vg-like protein levels, particularly in haemolymph, than animals from the other sampling sites. Interestingly, CI had high values in these polluted sites. In June only, Vg-like proteins and Ca²⁺ levels in haemolymph exhibited similar trends in both bivalve species at most sampling sites. The responsiveness of bivalves to environmental xenoestrogens was higher in June, allowing better discrimination among sites. The present study demonstrates that animals from highly polluted areas have increased Vg-like protein levels. As endocrine disruption due to exposure to estrogenic compounds may cause fertility reduction, alterations in the sex ratio, and a decrease in reproductive rate, a condition of potential risk for bivalve populations in estuarine areas is highlighted.     

First evidence of altered vitellogenin-like protein levels in clam Tapes philippinarum and in cockle Cerastoderma glaucum from the Lagoon of Venice

Possible xenoestrogenic effects were investigated, for the first time, in two bivalve species from the Lagoon of Venice (Italy): the clam Tapes philippinarum and the cockle Cerastoderma glaucum. Bivalves were collected far from their reproductive phase at the very early stage of gametogenesis (January), and in the pre-spawning period (June) in six sites. Vitellogenin (Vg)-like proteins (a biomarker of exposure to estrogenic compounds) were measured by the alkali-labile phosphate method (ALP), in both haemolymph and digestive gland from males and females (when it was possible to distinguish sex by microscopic observation of gonadal tissue). Haemolymph calcium concentrations (a parameter considered closely related to the presence of Vg-like proteins) and the bivalve condition index (CI) were also measured. In both seasons, bivalves collected at Campalto (near a sewage treatment plant) and Marghera (a highly contaminated area) had higher Vg-like protein levels, particularly in haemolymph, than animals from the other sampling sites. Interestingly, CI had high values in these polluted sites. In June only, Vg-like proteins and Ca²⁺ levels in haemolymph exhibited similar trends in both bivalve species at most sampling sites. The responsiveness of bivalves to environmental xenoestrogens was higher in June, allowing better discrimination among sites. The present study demonstrates that animals from highly polluted areas have increased Vg-like protein levels. As endocrine disruption due to exposure to estrogenic compounds may cause fertility reduction, alterations in the sex ratio, and a decrease in reproductive rate, a condition of potential risk for bivalve populations in estuarine areas is highlighted.     

Biomarker responses as indication of contaminant effects in blue mussel (Mytilus edulis) and female eelpout (Zoarces viviparus) from the southwestern Baltic Sea

During a field study performed in spring and autumn 2001 and 2002, blue mussels (Mytilus edulis) and female eelpout (Zoarces viviparus) were collected at three locations in the Wismar Bay (Baltic Sea), and several biomarkers of contaminant effects were analysed. Besides seasonal and inter-annual variations, biomarker signals were most pronounced at the location closest to Wismar Harbour (Wendorf) in both species. Lysosomal membrane stability (LMS) was lowest and acetylcholinesterase activity (AChE) was significantly reduced. Frequency of micronuclei (MN) was significantly higher (in blue mussels), indicating mutagenic effects. In eelpout elevated levels of DNA adducts, EROD induction and PAH-metabolites were measured. Metallothionein (MT), biomarker for trace metal exposure, showed a gradient only in spring. Organochlorine contaminant analyses (PCBs, DDTs) corresponded to the observed biomarker levels. The results obtained clearly demonstrate pollution effects in the southwestern Baltic Sea. Moreover, they show that a multibiomarker approach is also applicable in a brackish water environment.     

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.     

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.     

Histological Changes in a Few Tissues of Notopterus notopterus Induced by Sublethal and Lethal Concentrations of Pulp,Paper and Mixed Pulp and Paper Wastes

In the present work, histological changes in the gill, liver, kidney and intestine of Notopterus notopterus, induced by pulp waste, paper waste and mixed pulp and paper waste, have been studied both at sublethal and lethal concentrations. The pulp waste produces much more histological changes both at sublethal and lethal concentrations in the gill, liver, kidney and intestine as compared to paper waste and mixed pulp and paper waste. The changes are much more remarkable in the case of sublethal concentration, as compared to a lethal concentration. In a sublethal concentration, the localized necrosis, atrophy and partial and total liquefaction of epithelial cells, pilaster cells, mucous cells of the gill, hepatic cells of the liver, mesenchymatous cells, renal tubule cells and Bowman's capsule of the kidney, mucosal and submucosal cells and blood vessels of intestine have been observed. In lethal concentration, the changes are much more marked in the gill, as compared to the liver, kidney and intestine. The histological changes, produced in the different types of cells and in the tissue as a whole, can be correlated with the different chemical components present in the pulp waste, paper waste and mixed pulp and paper waste.