Bioassays, as an addition to physico-chemical water quality evaluation, can provide information on the toxic effects of pollutants present in the water. In this study, a broad evaluation of environmental health risks from industrial wastewater along the Yangtze River, China, was conducted using a battery of bioassays. Toxicity tests showed that the wastewater treatment processes were effective at lowering acetylcholinesterase (AChE) inhibition, HepG2 cells' cytotoxicity, the estrogenic effect in T47D-Kbluc cells, DNA damage of Euglena gracilis and the mutagenicity of Salmonella typhimurium in the analyzed wastewater samples. Polycyclic aromatic hydrocarbons (PAHs) were identified as potential major toxic chemicals of concern in the wastewater samples of W, J and T wastewater treatment plants; thus, the potential harm of PAHs to aquatic organisms has been investigated. Based on the health risk assessment model, the risk index of wastewater from the industrial parks along the Yangtze River was below one, indicating that the PAHs were less harmful to human health through skin contact or respiratory exposure. Overall, the biological toxicity tests used in this study provide a good basis for the health risk assessment of industrial wastewater and a scientific reference for the optimization and operation of the treatment process.

Mercury (Hg) is a metal with toxic effects on the environment, including living organisms. Organic Hg derivatives readily penetrate biological membranes and pose a particular health risk. Food of aquatic origin is the main source of human exposure to methylmercury (MeHg). In Poland, the consumption of fishery and aquaculture products has been gradually increasing. The aim of this study was to determine the content of Hg in fish intended for human consumption and purchased in Poland. The total Hg content of the edible parts of different species of marine and freshwater fish was analysed. The range of Hg content in all fish was 0.004-0.827 mg/kg, with an arithmetic mean of 0.084 mg/kg. The concentration of Hg in marine fish (0.100 mg/kg) was higher than in freshwater fish (0.063 mg/kg). The highest concentration of Hg was found in tuna. The Estimated Hazard Quotient (EHQ) calculated for the tuna samples analysed was >1. This may represent a potential health risk for consumers. The consumption of the other fish analysed was considered safe on the basis of the EHQ. The Hg content of the analysed fish samples did not exceed the current limits for food.

Pharmaceuticals and hormones (PhACs) enter the aquatic environment in multiple ways, posing potential adverse effects on non-target organisms. They have been widely detected in drinking water sources, challenging water companies to reassure good quality drinking water. The aim of this study was to evaluate the concentration of sixteen PhACs in both raw and treated drinking water sources in the Metropolitan Area of Turin-where Società Metropolitana Acque Torino (SMAT) is the company in charge of the water cycle management-and evaluate the potential human health risks associated to these compounds. Multivariate spatial statistical analysis techniques were used in order to characterize the areas at higher risk of pollution, taking into account the already existing SMAT sampling points' network. Health risks were assessed considering average detected concentrations and provisional guideline values for individual compounds as well as their combined mixture. As reported in the just-issued Drinking Water Directive 2020/2184/UE, in order to establish priority substances, a risk assessment of contaminants present in raw drinking water sources is required for monitoring, identifying potential health risks and, if necessary, managing their removal. The results showed negligibly low human health risks in both raw water sources and treated water.

Imidacloprid (IMI) is the first-generation neonicotinoid insecticide. But, the long-term use of IMI as a pesticide has caused severe water pollution. Recently, the toxicity of IMI to aquatic organisms has received increasing attention. This study aimed to investigate the absorption and distribution of IMI in various tissues (gills, intestine, liver, muscle, brain and gonads) of goldfish through short-term and continuous exposure tests over 28 days. The results of short-term exposure indicated that the concentration of IMI and its metabolites in tissues at the transfer stage decreased steadily after 1 day of 40 mg/L IMI water treatment and was below the detection limit after 3 days. Continuous exposure for 28 days at various treatment concentrations showed that the concentrations of IMI and its metabolites differed significantly among the different tissues of the goldfish. In the 20 mg/L treatment group (S1), the highest concentration of IMI was found in the liver (12.04 μg/gtissue), followed by the intestine (9.91 μg/gtissue), muscle (6.20 μg/gtissue), gill (6.11 μg/gtissue), gonads (5.22 μg/gtissue) and brain (2.87 μg/gtissue). In the 40 mg/L treatment group (S2), the order of the tissue concentrations was similar to that of the S1 group, with the highest concentration observed in the liver (12.04 μg/gtissue), followed by the intestine (9.91 μg/gtissue), muscle (6.20 μg/gtissue), gill (6.11 μg/gtissue), gonads (5.22 μg/gtissue) and brain (2.87 μg/gtissue). Furthermore, the study detected 5-hydroxy-IMI, IMI urea and 6-chloronicotinic acid in IMI metabolites in all tissues, while IMI was detected only in the intestine and liver. Overall, the results of this study contribute to a better understanding of the metabolic behavior of IMI in organisms and provide new data to support the assessment of IMI toxicity in fish.

Zinc oxide (ZnO) is the most efficient curing activator employed in the industrial rubber production. However, ZnO and Zn(II) ions are largely recognized as an environmental hazard being toxic to aquatic organisms, especially considering Zn(II) release during tire lifecycle. In this context, aiming at reducing the amount of microcrystalline ZnO, a novel activator was recently synthetized, constituted by ZnO nanoparticles (NPs) anchored to silica NPs (ZnO-NP@SiO2-NP). The objective of this work is to define the possible hazards deriving from the use of ZnO-NP@SiO2-NP compared to ZnO and SiO2 NPs traditionally used in the tire industry. The safety of the novel activators was assessed by in vitro testing, using human lung epithelial (A549) and immune (THP-1) cells, and by the in vivo model zebrafish (Danio rerio). The novel manufactured nanomaterial was characterized morphologically and structurally, and its effects evaluated in vitro by the measurement of the cell viability and the release of inflammatory mediators, while in vivo by the Fish Embryo Acute Toxicity (FET) test. Resulting data demonstrated that ZnO-NP@SiO2-NP, despite presenting some subtoxic events, exhibits the lack of acute effects both in vitro and in vivo, supporting the safe-by-design development of this novel material for the rubber industry.

Despite widespread industrial use, the environmental safety of hydroquinone (HQ), a benzene compound from plants used in processes like cosmetics, remains uncertain. This study evaluated the ecotoxicological impact of HQ on soil and river environments, utilizing non-target indicator organisms from diverse trophic levels: Daphnia magna, Aliivibrio fischeri, Allium cepa, and Eisenia fetida. For a more environmentally realistic assessment, microbial communities from a river and untreated soil underwent 16S rRNA gene sequencing, with growth and changes in community-level physiological profiling assessed using Biolog EcoPlate™ assays. The water indicator D. magna exhibited the highest sensitivity to HQ (EC50 = 0.142 µg/mL), followed by A. fischeri (EC50 = 1.446 µg/mL), and A. cepa (LC50 = 7.631 µg/mL), while E. fetida showed the highest resistance (EC50 = 234 mg/Kg). Remarkably, microbial communities mitigated HQ impact in both aquatic and terrestrial environments. River microorganisms displayed minimal inhibition, except for a significant reduction in polymer metabolism at the highest concentration (100 µg/mL). Soil communities demonstrated resilience up to 100 µg/mL, beyond which there was a significant decrease in population growth and the capacity to metabolize carbohydrates and polymers. Despite microbial mitigation, HQ remains highly toxic to various trophic levels, emphasizing the necessity for environmental regulations.

The bioconcentration factor (BCF) is one of the metrics used to evaluate the potential of a substance to bioaccumulate into aquatic organisms. In this work, linear and non-linear regression QSARs were developed for the prediction of log BCF using different computational approaches, and starting from a large and structurally heterogeneous dataset. The new MLR-OLS and ANN regression models have good fitting with R2 values of 0.62 and 0.70, respectively, and comparable external predictivity with R2ext 0.64 and 0.65 (RMSEext of 0.78 and 0.76), respectively. Furthermore, linear and non-linear classification models were developed using the regulatory threshold BCF >2000. A class balanced subset was used to develop classification models which were applied to chemicals not used to create the QSARs. These classification models are characterized by external and internal accuracy up to 84% and 90%, respectively, and sensitivity and specificity up to 90% and 80%, respectively. QSARs presented in this work are validated according to regulatory requirements and their quality is in line with other tools available for the same endpoint and dataset, with the advantage of low complexity and easy application through the software QSAR-ME Profiler. These QSARs can be used as alternatives for, or in combination with, existing models to support bioaccumulation assessment procedures.

Organophosphate pesticides (OPs) are toxic substances that contaminate aquatic environments, interfere with the development of the nervous system, and induce Neurodevelopmental Toxicity (NDT) in animals and humans. The canonical mechanism of OP neurotoxicity involves the inhibition of acetylcholinesterase (AChE), but other mechanisms non-AChE are also involved and not fully understood. We used network toxicology and molecular docking to identify molecular targets and toxicity mechanisms common to OPs. Targets related to diazinon-oxon, chlorpyrifos oxon, and paraoxon OPs were predicted using the Swiss Target Prediction and PharmMapper databases. Targets related to NDT were compiled from GeneCards and OMIM databases. In order to construct the protein-protein interaction (PPI) network, the common targets between OPs and NDT were imported into the STRING. Network topological analyses identified EGFR, MET, HSP90AA1, and SRC as hub nodes common to the three OPs. Using the Reactome pathway and gene ontology, we found that signal transduction, axon guidance, cellular responses to stress, and glutamatergic signaling activation play key roles in OP-induced NDT.

Pharmaceuticals pose a great threat to organisms inhabiting the aquatic environment. Non-steroidal anti-inflammatory drugs (NSAIDs) are major pharmaceutical pollutants with a significant presence in freshwater ecosystems. In this study, the impact of indomethacin and ibuprofen, two of the most commonly prescribed NSAIDs, was assessed on Daphnia magna. Toxicity was assessed as the immobilization of animals and used to determine non-lethal exposure concentrations. Feeding was assessed as a phenotypic endpoint and key enzymes were used as molecular endpoints of physiology. Feeding was decreased in mixture exposures for five-day-old daphnids and neonates. Furthermore, animals were exposed to NSAIDs and their mixture in chronic and transgenerational scenarios revealing changes in key enzyme activities. Alkaline and acid phosphatases, lipase, peptidase, β-galactosidase, and glutathione-S-transferase were shown to have significant changes in the first generation at the first and third week of exposure, and these were enhanced in the second generation. On the other hand, the third recovery generation did not exhibit these changes, and animals were able to recover from the induced changes and revert back to the control levels. Overall, our study points towards transgenerational exposures as more impactful laboratory studies to understand pharmaceutical stressors with a combination of molecular and phenotypic markers of physiology.

Dinoflagellates of the genus Amphidinium can produce a variety of polyketides, such as amphidinols (AMs), amphidinoketides, and amphidinin, that have hemolytic, cytotoxic, and fish mortality properties. AMs pose a significant threat to ecological function due to their membrane-disrupting and permeabilizing properties, as well as their hydrophobicity. Our research aims to investigate the disparate distribution of AMs between intracellular and extracellular environments, as well as the threat that AMs pose to aquatic organisms. As a result, AMs containing sulphate groups such as AM19 with lower bioactivity comprised the majority of A. carterae strain GY-H35, while AMs without sulphate groups such as AM18 with higher bioactivity displayed a higher proportion and hemolytic activity in the extracellular environment, suggesting that AMs may serve as allelochemicals. When the concentration of extracellular crude extracts of AMs reached 0.81 µg/mL in the solution, significant differences in zebrafish embryonic mortality and malformation were observed. Over 96 hpf, 0.25 μL/mL of AMs could cause significant pericardial edema, heart rate decrease, pectoral fin deformation, and spinal deformation in zebrafish larvae. Our findings emphasized the necessity of conducting systematic research on the differences between the intracellular and extracellular distribution of toxins to gain a more accurate understanding of their effects on humans and the environment.

Pharmaceuticals such as antidepressants are designed to be bioactive at low concentrations. According to their mode of action, they can also influence non-target organisms due to the phylogenetic conservation of molecular targets. In addition to the pollution by environmental chemicals, the topic of microplastics (MP) in the aquatic environment came into the focus of scientific and public interest. The aim of the present study was to investigate the influence of the antidepressant amitriptyline in the presence and absence of irregularly shaped polystyrene MP as well as the effects of MP alone on juvenile brown trout (Salmo trutta f. fario). Fish were exposed to different concentrations of amitriptyline (nominal concentrations between 1 and 1000 µg/L) and two concentrations of MP (104 and 105 particles/L; <50 µm) for three weeks. Tissue cortisol concentration, oxidative stress, and the activity of two carboxylesterases and of acetylcholinesterase were assessed. Furthermore, the swimming behavior was analyzed in situations with different stress levels. Exposure to amitriptyline altered the behavior and increased the activity of acetylcholinesterase. Moreover, nominal amitriptyline concentrations above 300 µg/L caused severe acute adverse effects in fish. MP alone did not affect any of the investigated endpoints. Co-exposure caused largely similar effects such as the exposure to solely amitriptyline. However, the effect of amitriptyline on the swimming behavior during the experiment was alleviated by the higher MP concentration.

Methylene blue (MB) is a disinfectant used in aquaculture to prevent and treat fish diseases. However, the release of MB can pose a risk to the receiving water bodies. Zooplankton are the most sensitive organisms among aquatic life. Hence, this study examined the acute and chronic toxic effects of MB on zooplankton using Daphnia magna (D. magna) as a test organism to provide basic data for risk assessment. The results show that 48 h-EC50 and 24 h-LC50 were 61.5 ± 2.3 and 149.0 ± 2.2 μg/L, respectively. Chronic exposure to MB affected the heart rate, beat frequency of the thoracic limbs, and reproductive ability of D. magna at environmental concentrations higher than 4.7 μg/L. The cumulative molts, time to production of the first brood, and total number of living offspring were affected at different MB concentrations, while "abortions" were observed in high-exposure groups. The activity of superoxide dismutase was increased, while glutathione S-transferase activity was stimulated at low concentrations and inhibited at high concentrations. In addition, the malondialdehyde content increased with increasing concentrations of MB. Our findings demonstrate the impact of MB on the reproduction and growth of freshwater species, as well as their physiological responses. These results have implications for establishing guidelines on the use of MB in aquaculture and setting discharge standards.

The presence of plastic cosmetic microbeads in the environment due to their extensive use in society and inevitable dispersal into wastewater is concerning. Therefore, it is vital to understand the processes of microplastic uptake and elimination by aquatic organisms, and to further assess their potential to cause harmful effects and wider impacts. We therefore investigated the short-term (48-h) and long-term (21-d) uptake, elimination, and effects of exposure to polyethylene microbeads (a mixture of fragments and spheres extracted from commercially available facial scrubs) on the freshwater snail, Biomphalaria glabrata. We found fast uptake in the short-term (75 μg/g/h) and the long-term (6.94 μg/g/h) in B. glabrata exposed to 800 particles/200-mL and 80 particles/200-mL, respectively. Irregular fragments were more easily ingested and egested compared to spheres (ANOVA, p < 0.05) in both 48-h and 21-d exposures. The mean size of the fragments in B. glabrata tissues (413 ± 16 μm) after 48-h exposure was significantly larger than that of the standard sample (369 ± 26 μm) (ANOVA, F3,20 = 3.339, p = 0.033), suggesting that aggregation in the gut may occur. Floating feces containing microbeads were observed in the long-term exposure, which could alter the fate, behavior, and bioavailability of egested microbeads. No significant effects on survival and growth were shown within 48-h or 21-d exposure periods. Thus, further studies on the specific features of microplastics (e.g., their shape and size) influencing uptake and elimination, as well as toxic molecular mechanisms, should be explored in future ecotoxicological studies.

The aim of this study was to understand the distribution of the personal care products nonylphenol (NP), triclosan (TCS), benzophenone-3 (BP-3), and caffeine in the sludges from three wastewater treatment plants (WWTP-A, -B, and -C) in southern Taiwan. The four compounds were analyzed from activated sludge and dewatered sludge samples, and then the samples were treated with pressure-assisted ozonation under different conditions and removal efficiencies. All four target compounds were detected, especially NP, which was detected in the highest concentrations in the activated sludges of WWTP-A and dewatered sludges of WWTP-C at 17.19 ± 4.10 and 2.41 ± 1.93 µg/g, respectively. TCS was dominant in dewatered sludges from WWTP-B, and the highest detected concentration was 13.29 ± 6.36 µg/g. Removals of 70% and 90% were attained under 150 psi at 40 cycles for NP and TCS, respectively, with 5 min of ozonation reaction time, a solid/water ratio of 1:20, and 2% ozone concentration. Ecological risk quotients (RQs) were calculated by the ratios of the 10-day Hyalella azteca (freshwater amphipod) LC50 to the environmental concentrations of the target compounds. High RQs were found to be >10 for NP, TCS, and BP-3 in untreated sludges, resulting in significant ecological risks to aquatic organisms when the sludges are arbitrarily disposed. However, the toxic effects on Hyalella azteca were not significantly different among ozone sludge treatments. The reason for this may be related to the formation of toxic oxidation by-products and incomplete mineralization of organic compounds. This could also be true for unknown intermediates. The relatively high detection frequencies of these emerging compounds in WWTP sludges requires further applications and treatments.

Xenobiotics released in the environment can be taken up by aquatic and terrestrial organisms and can accumulate at higher concentrations through the trophic chain. Bioaccumulation is therefore one of the PBT properties that authorities require to assess for the evaluation of the risks that chemicals may pose to humans and the environment. The use of an integrated testing strategy (ITS) and the use of multiple sources of information are strongly encouraged by authorities in order to maximize the information available and reduce testing costs. Moreover, considering the increasing demand for development and the application of new approaches and alternatives to animal testing, the development of in silico cost-effective tools such as QSAR models becomes increasingly important. In this study, a large and curated literature database of fish laboratory-based values of dietary biomagnification factor (BMF) was used to create externally validated QSARs. The quality categories (high, medium, low) available in the database were used to extract reliable data to train and validate the models, and to further address the uncertainty in low-quality data. This procedure was useful for highlighting problematic compounds for which additional experimental effort would be required, such as siloxanes, highly brominated and chlorinated compounds. Two models were suggested as final outputs in this study, one based on good-quality data and the other developed on a larger dataset of consistent Log BMFL values, which included lower-quality data. The models had similar predictive ability; however, the second model had a larger applicability domain. These QSARs were based on simple MLR equations that could easily be applied for the predictions of dietary BMFL in fish, and support bioaccumulation assessment procedures at the regulatory level. To ease the application and dissemination of these QSARs, they were included with technical documentation (as QMRF Reports) in the QSAR-ME Profiler software for QSAR predictions available online.

Microplastics (MPs)' ingestion has been demonstrated in several aquatic organisms. This process may facilitate the hydrophobic waterborne pollutants or chemical additives transfer to biota. In the present study the suitability of a battery of biomarkers on oxidative stress, physiology, tissue function and metabolic profile was investigated for the early detection of adverse effects of 21-day exposure to polystyrene microplastics (PS-MPs, sized 5-12 μm) in the liver and gills of zebrafish Danio rerio and perch, Perca fluviatilis, both of which are freshwater fish species. An optical volume map representation of the zebrafish gill by Raman spectroscopy depicted 5 μm diameter PS-MP dispersed in the gill tissue. Concentrations of PS-MPs close to the EC50 of each fish affected fish physiology in all tissues studied. Increased levels of biomarkers of oxidative damage in exposed fish in relation to controls were observed, as well as activation of apoptosis and autophagy processes. Malondialdehyde (MDA), protein carbonyls and DNA damage responses differed with regard to the sensitivity of each tissue of each fish. In the toxicity cascade gills seemed to be more liable to respond to PS-MPs than liver for the majority of the parameters measured. DNA damage was the most susceptible biomarker exhibiting greater response in the liver of both species. The interaction between MPs and cellular components provoked metabolic alterations in the tissues studied, affecting mainly amino acids, nitrogen and energy metabolism. Toxicity was species and tissue specific, with specific biomarkers responding differently in gills and in liver. The fish species that seemed to be more susceptible to MPs at the conditions studied, was P. fluviatilis compared to D. rerio. The current findings add to a holistic approach for the identification of small sized PS-MPs' biological effects in fish, thus aiming to provide evidence regarding PS-MPs' environmental impact on wild fish populations and food safety and adequacy.

Microplastics (MPs) are detected in the water, sediments, as well as biota, mainly as a consequence of the degradation of plastic products/waste under environmental conditions. Due to their potentially harmful effects on ecosystems and organisms, MPs are regarded as emerging pollutants. The highly problematic aspect of MPs is their interaction with organic and inorganic pollutants; MPs can act as vectors for their further transport in the environment. The objective of this study was to investigate the effects of ageing on the changes in physicochemical properties and size distribution of polyethylene terephthalate (PET), as well as to investigate the adsorption capacity of pristine and aged PET MPs, using pharmaceutical diclofenac (DCF) as a model organic pollutant. An ecotoxicity assessment of such samples was performed. Characterization of the PET samples (bottles and films) was carried out to detect the thermooxidative aging effects. The influence of the temperature and MP dosage on the extent of adsorption of DCF was elucidated by employing an empirical modeling approach using the response surface methodology (RSM). Aquatic toxicity was investigated by examining the green microalgae Pseudokirchneriella subcapitata. It was found that the thermooxidative ageing process resulted in mild surface changes in PET MPs, which were reflected in changes in hydrophobicity, the amount of amorphous phase, and the particle size distribution. The fractions of the particle size distribution in the range 100-500 μm for aged PET are higher due to the increase in amorphous phase. The proposed mechanisms of interactions between DCF and PET MPs are hydrophobic and π-π interactions as well as hydrogen bonding. RSM revealed that the adsorption favors low temperatures and low dosages of MP. The combination of MPs and DCF exhibited higher toxicity than the individual components.

There is a growing concern that neuroactive chemicals released into the environment can perturb wildlife behaviour. Among these chemicals, pharmaceuticals such as antidepressants and anxiolytics have been receiving increasing attention, as they are specifically prescribed to modify behavioural responses. Many laboratory studies have demonstrated that some of these compounds can affect various aspects of the behaviour of a range of aquatic organisms; however, these investigations are focused on a very small set of neuroactive pharmaceuticals, and they often consider one compound at a time. In this study, to better understand the environmental and toxicological dimension of the problem, we considered all pharmaceuticals explicitly intended to modulate the central nervous system (CNS), and we hypothesised that these compounds have higher probability of perturbing animal behaviour. Based on this hypothesis, we used the classification of pharmaceuticals provided by the British National Formulary (based on their clinical applications) and identified 210 different CNS-acting pharmaceuticals prescribed in the UK to treat a variety of CNS-related conditions, including mental health and sleep disorders, dementia, epilepsy, nausea, and pain. The analysis of existing databases revealed that 84 of these compounds were already detected in surface waters worldwide. Using a biological read-across approach based on the extrapolation of clinical data, we predicted that the concentration of 32 of these neuroactive pharmaceuticals in surface waters in England may be high enough to elicit pharmacological effects in wild fish. The ecotoxicological effects of the vast majority of these compounds are currently uncharacterised. Overall, these results highlight the importance of addressing this environmental challenge from a mixture toxicology and systems perspective. The knowledge platform developed in the present study can guide future region-specific prioritisation efforts, inform the design of mixture studies, and foster interdisciplinary efforts aimed at identifying novel approaches to predict and interpret the ecological implications of chemical-induced behaviour disruption.

Pb is one of the most extensively used harmful heavy metals in Bangladesh, and its occurrence in waters affects aquatic organisms significantly. The tropical pearl mussel, Lamellidens marginalis, was exposed to different concentrations (T1 21.93 mgL-1, T2 43.86 mgL-1, and T3 87.72 mgL-1) of Pb(NO3)2 and was evaluated against a control C 0 mgL-1 of Pb(NO3)2, followed by a 96 h acute toxicity test. The LC50 value was recorded as 219.32 mgL-1. The physicochemical parameters were documented regularly for each treatment unit. The values of % SGR, shell weight, soft tissue wet weight, and weight gain remained statistically higher for the control group in comparison with the treatment. No mortality was noted for control units, while a gradually decreased survival rate was recorded for the different treatment groups. Fulton's condition factor was recorded as highest in the control and lowest in the T3 unit, while the condition indices did not vary between the control and treatment groups. The hemocyte was accounted as maximum in the control and T1, while minimum in T2 and T3. The serum lysosomal parameters also followed a similar pattern, and a significantly low level of lysosomal membrane stability, and serum lysosome activity was noted for T3 and T2 units in comparison to the control group. The histology of the gill, kidney, and muscle was well structured in the control group, while distinct pathologies were observed in the gill, kidney, and muscle tissue of different treatment groups. The quantitative comparison revealed that the intensity of pathological alteration increased as the dosage of Pb increased. The current study, therefore, indicated that intrusion of Pb(NO3)2 in the living medium significantly alters growth performance and hemocyte counts, and chronic toxicity induces histomorphological abnormalities in vital organs.