Toxic Metal Pollution in Pakistan and Its Possible Risks to Public Health.

Environmental pollution has increased many folds in recent years and in some places has reached levels that are toxic to living things. Among pollutant types, toxic heavy metals and metalloids are among the chemicals that pose the highest threat to biological systems (Jjemba 2004). Unlike organic pollutants, which are biodegradable, heavy metals are not degraded into less hazardous end products (Gupta et al. 2001). Low concentrations of some heavy metals are essential for life, but some of them like Hg, As, Pb and Cd are biologically non-essential and very toxic to living organisms. Even the essential metals may become toxic if they are present at a concentration above the permissible level (Puttaiah and Kiran 2008). For example, exposure to Zn and Fe oxides produce gastric disorder and vomiting, irritation of the skin and mucous membranes. Intake of Ni, Cr, Pb, Cd and Cu causes heart problems, leukemia and cancer, while Co and Mg can cause anemia and hypertension (Drasch et al. 2006). Similarly, various studies indicated that overexposure to heavy metals in air can cause cardiovascular disorders (Miller et al. 2007; Schwartz 2001), asthma (Wiwatanadate and Liwsrisakun 2011), bronchitis/emphysema (Pope 2000), and other respiratory diseases (Dominici et al. 2006).

Effects of industrial wastewater on growth and biomass production in commonly grown vegetables.

In developing countries like Pakistan, irrigation of crops with industrial and municipal wastewater is a common practice. However, the impact of wastewater irrigation on vegetables growth has rarely been studied. Therefore, the present study was conducted to determine the effect of industrial wastewater on the germination and seedling growth of some commonly grown vegetables in Pakistan. Wastewater samples were collected from two different industries (marble industry and match alam factory) at Hayatabad Industrial Estate (HIE) in Peshawar, Pakistan, and their effect on different growth parameters of four vegetables including Hibiscus esculentus, Lactuca sativa, Cucumis sativus, and Cucumis melo was investigated. The obtained results revealed that wastewater from marble industry did not affect seed germination except a minor inhibition in H. esculentus. Effluents from match alam factory stimulated seed germination in C. melo and C. sativus but had no effect on seed germination in the other two vegetables. Wastewater increased root and shoot length in H. esculentus, L. sativa and C. melo, but decreased it in C. sativus. Similarly, differential effects of wastewater were observed on fresh and dry biomass of seedlings in all vegetables. It can be concluded that wastewater may have different effects on different crops, depending upon the nature of wastewater and sensitivity of a plant species to wastewater.

Assessment of the impact of chlorophyll derivatives to control parasites in aquatic ecosystems.

Several research groups have studied new biopesticides which are less toxic to the environment and capable of controlling the vectors of parasitic diseases, especially in aquatic ecosystems. Pest control by photodynamic substances is an alternative to chemical or other measures, with chlorophyll and its derivatives as the most studied substances supported by their easy availability and low production costs. The impact of chlorophyll derivatives on four different species, a small crustacean (Daphnia similis), a unicellular alga (Euglena gracilis) and two species of fish (Astyanax bimaculatus and Cyprynus carpio) were tested under short-term conditions. In addition, the effects of long-term exposure were evaluated in D. similis and E. gracilis. In short-term tests, mortality of D. similis (EC50 = 7.75 mg/L) was most strongly affected by chlorophyllin, followed by E. gracilis (EC50 = 12.73 mg/L). The fish species showed a greater resistance documented by their EC50 values of 17.58 and 29.96 mg/L in C. carpio and A. bimaculatus, respectively. A risk quotient is calculated by dividing an estimate of exposure by an estimate of effect. It indicated that chlorophyll derivatives can be applied in nature to control the vectors of parasitic diseases under short-term conditions, but long-term exposure requires new formulations.

Comparative toxicity of physiological and biochemical parameters in Euglena gracilis to short-term exposure to potassium sorbate.

Potassium sorbate is the potassium salt of sorbic acid, is a widespread and efficient antioxidant that has multiple functions in plants, traditionally associated with the reactions of photosynthesis; however, it has moderate toxicity to various species including rat, fish, bacteria and human health. The effects of potassium sorbate on the movement and photosynthetic parameters of Euglena gracilis were studied during short-term exposure. Potassium sorbate showed acute toxicity to the green flagellate E. gracilis affecting different physiological parameters used as endpoints in an automatic bioassay such as motility, precision of gravitational orientation (r-value), upward movement and alignment, with mean EC50 values of 2867.2 mg L(-1). The concentrations above 625 mg L(-1) of potassium sorbate induce an inhibition of the photosynthetic efficiency and electron transport rate and, in concentrations more than 2500.0 mg L(-1), the Euglena cells undergo a complete inhibition of photosynthesis even at low light irradiation.

Chlorophyll derivatives can be an efficient weapon in the fight against dengue.

Dengue, a mosquito-borne viral infection, is one of the major public health concerns in the tropical and subtropical regions of the world. Approximately, 2.5 billion people across the world are at risk from dengue and 50 to 100 million new infections of dengue occur annually. There is yet no vaccine or medicine available against dengue, and treatment remains only supportive. Targeting its vector by a combination of biological and chemical approaches and management of breeding sites are currently the only existing approaches to control or eliminate dengue. Chlorophyll derivatives like chlorophyllin and pheophorbide have been reported as effective natural photosensitizers against larvae of several insects including flies. Chlorophyll derivatives were also reported effective against larval stages of freshwater snails as well as against certain parasites of fish. This article briefly discusses the possible application of chlorophyll derivatives in controlling dengue vectors and hence the disease itself. Chlorophyll derivatives can prove to be a good contributor in an integrated approach against dengue.

Potential environmental toxicity from hemodialysis effluent.

Understanding the toxicity of certain potentially toxic compounds on various aquatic organisms allows to assess the impact that these pollutants on the aquatic biota. One source of pollution is the wastewater from hemodialysis. The process of sewage treatment is inefficient in inhibition and removal of pathogenic bacteria resistant to antibiotics in this wastewater. In many countries, such as Brazil, during emergencies, sewage and effluents from hospitals are often dumped directly into waterways without any previous treatment. The objective of this study was to characterize the effluents generated by hemodialysis and to assess the degree of acute and chronic environmental toxicity. The effluents of hemodialysis showed high concentrations of nitrites, phosphates, sulfates, ammonia, and total nitrogen, as well as elevated conductivity, turbidity, salinity, biochemical and chemical oxygen demand, exceeding the thresholds defined in the CONAMA Resolution 430. The samples showed acute toxicity to the green flagellate Euglena gracilis affecting different physiological parameters used as endpoints in an automatic bioassay such as motility, precision of gravitational orientation (r-value), compactness, upward movement, and alignment, with mean EC50 values of recalculate as 76.90 percent (±4.68 percent) of the undiluted effluents. In tests with Daphnia magna, the acute toxicity EC50 was 86.91 percent (±0.39 percent) and a NOEC value of 72.97 percent and a LEOC value 94.66 percent.

Ecotoxicity evaluation of a liquid detergent using the automatic biotest ECOTOX.

Synthetic detergents are common pollutants reaching aquatic environments in different ways after usage at homes, institutions and industries. In this study a liquid detergent, used for dish washing, was evaluated for its toxicity during long- and short-term tests using the automatic biotest ECOTOX. Different parameters of Euglena gracilis like motility, swimming velocity, gravitactic orientation, cell compactness and cell growth were used as end points. In short-term experiments, the maximum adverse effects on motility, velocity, cell shape and gravitaxis were observed after 1 h of exposure. With further increase in exposure time to the detergent a slight recovery of these parameters was observed. In long-term experiments, the detergent caused severe disturbances to E. gracilis. Motility, cell growth and cell compactness (shape) with EC50 values of 0.064, 0.18 and 2.05 %, respectively, were found as the most sensitive parameters to detergent stress. There was a slight positive effect on gravitactic orientation at the lowest two concentrations; at higher concentrations of the detergent cells orientation was highly impaired giving EC50 values of 1.75 and 2.52 % for upward swimming and r-value, respectively.

Chronic toxicity of a laundry detergent to the freshwater flagellate Euglena gracilis.

Chronic toxicity of the common laundry detergent Ariel on the freshwater alga Euglena gracilis was investigated by growing the alga in a medium containing the detergent for 7 days. Cell density, motility, swimming velocity, gravitactic orientation, cell shape, photosynthesis and concentration of light-harvesting pigments were used as end point parameters for the assessment of toxicity. Cell density was significantly reduced at a concentration of 1 mg l(-1) or above. Among the other tested parameters, with the exception of cell shape, gravitaxis and chlorophyll b, all were adversely affected by the detergent at concentrations exceeding 1 mg l(-1). It is concluded that long-term (7-days) exposure to the detergent caused significant toxicity to E. gracilis. Furthermore, long-term tests with E. gracilis can be used as sensitive indicator for the toxicity assessment of laundry detergents in aquatic environments.

Optimizing conditions for the use of chlorophyll derivatives for photodynamic control of parasites in aquatic ecosystems.

Recently, it was demonstrated that mosquito larvae can be killed by means of photodynamic processes after the larvae have incorporated the photosensitizer chlorophyllin or pheophorbid, and were treated with light. The water-soluble substances were applied to and incorporated by the larvae in darkness. With Chaoborus sp. a dark incubation of about 3 h is sufficient to yield mortality of about 90% and ≥6 h resulted in almost 100% mortality during subsequent illumination. Temperature did not influence mortality of the larvae significantly in a treatment of 6 h dark incubation and subsequent 3 h illumination. At 10°C, 20°C, or 30°C, between 80% and 100% of the treated larvae died when the light intensity from a solar simulator was above 30 W/m(2). Lower irradiances were less effective. The LD(50) value of magnesium chlorophyllin was about 22.25 mg/l and for Zn chlorophyll 17.53 mg/l, while Cu chlorophyll (LD(50) 0.1 mg/l) was shown to be toxic also without light. Chlorophyllin, which was lyophilized immediately after extraction, was far more lethal to the larvae (LD(50) 14.88 mg/l) than air-dried Mg chlorophyllin.

Comparative toxicity of the pesticides carbofuran and malathion to the freshwater flagellate Euglena gracilis.

Pesticides are toxic chemicals used for agricultural as well as non-agricultural purposes. The toxicity of pesticides does not remain limited to the site of application but they also cause toxicity to non-target organisms in terrestrial as well as in aquatic environments. This study discusses the comparative toxicity of a carbamate (carbofuran) and an organophosphorus (malathion) pesticide to the freshwater flagellate Euglena gracilis during short- and long-term exposures. To evaluate the toxicity of the pesticides, different parameters of the flagellate, like cell density, motility, swimming velocity, cell shape, gravitactic orientation, photosynthetic efficiency, and concentration of light harvesting pigments, were used as end points. Carbofuran was found to be more toxic to E. gracilis than malathion and adversely affected almost all the tested parameters in short- and long-term experiments. The only significant adverse effect by malathion could be demonstrated on the swimming velocity of cells in short-term experiments. The adverse effects of the pesticides were more pronounced during short-term than during long-term exposure.

Molecular characterization of a calmodulin involved in the signal transduction chain of gravitaxis in Euglena gracilis.

The unicellular flagellate Euglena gracilis shows a negative gravitactic behavior. This is based on physiological mechanisms which in the past have been indirectly assessed. Meanwhile, it was possible to isolate genes involved in the signal transduction chain of gravitaxis. The DNA sequences of five calmodulins were found in Euglena, one of which was only known in its protein structure (CaM.1); the other four are new. The biosynthesis of the corresponding proteins of CaM.1-CaM.5 was inhibited by means of RNA interference to determine their involvement in the gravitactic signal transduction chain. RNAi of CaM.1 inhibits free swimming of the cells and pronounced cell-form aberrations. The division of cells was also hampered. After recovery from RNAi the cell showed precise negative gravitaxis again. Blockage of CaM.3 to CaM. 5 did not impair gravitaxis. In contrast, the blockage of CaM.2 has only a transient and not pronounced influence on motility and cell form, but leads to a total loss of gravitactic orientation for more than 30 days. This indicates that CaM.2 is an element in the signal transduction chain of gravitaxis in E. gracilis. The results are discussed with regard to the current working model of gravitaxis in E. gracilis.

Complementary UV-absorption of mycosporine-like amino acids and scytonemin is responsible for the UV-insensitivity of photosynthesis in Nostoc flagelliforme.

Mycosporine-like amino acids (MAAs) and scytonemin are UV-screening compounds that have presumably appeared early in the history of life and are widespread in cyanobacteria. Natural colonies of the UV-insensitive Nostoc flagelliforme were found to be especially rich in MAAs (32.1 mg g DW(-1)), concentrated in the glycan sheath together with scytonemin. MAAs are present in the form of oligosaccharide-linked molecules. Photosystem II activity, measured using PAM fluorescence and oxygen evolution, was used as a most sensitive physiological parameter to analyse the effectiveness of UV-protection. Laboratory experiments were performed under controlled conditions with a simulated solar radiation specifically deprived of UV-wavebands with cut-off filters (295, 305, 320, 345 and 395 nm). The UV-insensitivity of N. flagelliforme was found to cover the whole UV-A (315-400 nm) and UV-B (280-320 nm) range and is almost certainly due to the complementary UV-absorption of MAAs and scytonemin. The experimental approach used is proposed to be suitable for the comparison of the UV-protection ability in organisms that differ in their complement of UV-sunscreen compounds. Furthermore, this study performed with a genuinely terrestrial organism points to the relevance of marine photoprotective compounds for life on Earth, especially for the colonization of terrestrial environments.

Monitoring of Solar Irradiance in the High Andes.

Solar radiation has been measured in the high Andes near Laguna Lejia (latitude 23° 26' 23.30" S, longitude 67° 38' 14.29" W) at an elevation of 4715 m between December 2016 and December 2017. Irradiances were monitored in four wavelength channels: PAR (400-700 nm), UV-A (315-400 nm), UV-B (295-315 nm) and short-wavelength UV-B (295-310 nm) with a new radiometer. In addition, ambient temperatures were recorded. Record values have been found for PAR (exceeding 600 W m-2 ), UV-A (close to 95 W m-2 ), UV-B (3.13 W m-2 ) and short-wavelength UV-B (0.144 W m-2 ) during Austral spring. The winter irradiance values slightly exceeded 50% of these values. Maximal cloud effects due to multiple reflections were 45, 38, 32 and 35% higher than values under cloudless skies for PAR, UV-A, UV-B and short-wavelength UV-B, respectively. Record irradiance for this site shows a UV index reaching and exceeding 20, which is due to low solar zenith angles, the altitude, low water vapor and aerosol concentrations in the atmosphere as well as low total column ozone concentrations.

ELDONET--a decade of monitoring solar radiation on five continents.

The European light dosimeter network (ELDONET) comprises more than 40 stations in 24 countries on 5 continents. The present report compares solar radiation data in the photosynthetic active radiation, UV-A (315-400 nm) and UV-B (280-315 nm) wavelength ranges for 17 stations at different latitudes on the northern and southern hemispheres for up to 10 years of monitoring. While the maximal irradiances on clear days follow a latitudinal gradient due to the cosine dependence on the solar angle, the total doses strongly depend on the local climate and atmospheric conditions as well as the day-length distribution over the year. UV-B irradiances and doses are strongly influenced by the total column ozone, which is recorded for all covered stations.

Heterologous expression of photoactivated adenylyl cyclase (PAC) genes from the flagellate Euglena gracilis in insect cells.

The unicellular, green flagellate wild-type Euglena gracilis (strain Z) possesses two genes of the photoactivated adenylyl cyclase (PAC) family. The corresponding gene products were found to be responsible for step-up (but not step-down) photophobic responses as well as both positive and negative phototaxis. The proteins consist of two PACalpha (Mr 105 kDa) and two PACbeta (90 kDa) subunits. In an effort to produce sufficient amounts of PAC proteins, several routes of over-expression have been tried including homologous expression in Euglena and heterologous expression in Escherichia coli. All these approaches were hampered by low yield or formation of inclusion bodies. Therefore we decided to attempt a heterologous expression in an insect cell line. PACalpha and PACbeta were separately cloned in the transfer vector pBacPAK9 with a His tag attached. The transfer vector was subsequently cotransfected via baculovirus into the insect cells and amplified. For the expression both recombinant viruses (containing PACbeta and PACbeta, respectively) were cotransfected simultaneously into insect cells. The expressed proteins were analyzed in Western blots using PACalpha and PACbeta antibodies. Most of the proteins were found to be in soluble form in high yield. The recombinant PAC proteins were purified via their attached His tag on an anti-His resin. Adenylyl cyclase activity was quantified after blue-light excitation using a cAMP enzyme immunoassay kit.

Screening for unicellular algae as possible bioassay organisms for monitoring marine water samples.

ECOTOX is an automatic early warning system to monitor potential pollution of freshwater, municipal or industrial waste waters or aquatic ecosystems. It is based on a real time image analysis of the motility and orientation parameters of the unicellular, photosynthetic flagellate Euglena gracilis. In order to widen the use of the device to marine habitats and saline waters nine marine flagellates were evaluated as putative bioassay organisms, viz. Dunaliella salina, Dunaliella viridis, Dunaliella bardawil, Prorocentrum minimum Kattegat, P. minimum Lissabon, Tetraselmis suecica, Heterocapsa triquetra, Gyrodinium dorsum and Cryptomonas maculata. Because of their slow growth the last three strains were excluded from further evaluation. Selection criteria were ease of culture, density of cell suspension, stability of motility and gravitactic orientation. The sensitivity toward toxins was tested using copper(II) ions. The instrument allows the user to automatically determine effect-concentration (EC) curves from which the EC(50) values can be calculated. For the interpretation of the EC curves a sigmoid logistic model was proposed which proved to be satisfactory for all tested strains. The inhibition of the motility was considered as the most appropriate movement parameter as an endpoint. The Dunaliella species had the lowest sensitivity to copper with EC(50) values of 220, 198 and 176 mg/L for D. salina, D. bardawil and D. viridis, respectively, followed by T. suecica with an EC(50) value of 40 mg/L. The Prorocentrum species were found to be the most sensitive with an EC(50) value of 13.5 mg/L for P. minimum Lissabon and 7.5 mg/L for P. minimum Kattegat.

The European Light Dosimeter Network: four years of measurements.

The European Light Dosimeter Network (ELDONET) has now been functional for more than four years. The network is based on dosimeters which measure radiation in three biologically relevant wavelength bands (UV-B, 280-315 nm; UV-A, 315-400 nm; and Photosynthetic Active Radiation, PAR, 400-700 nm). The ELDONET network is currently based on 33 stations with 40 instruments. The distribution of the instruments all over Europe allows measurement of the latitudinal and longitudinal light climate distribution. In addition, several instruments are active in South America, New Zealand, India, Africa and Japan. With some exceptions, the measured yearly doses depend on the latitude. While the maximal daily doses are almost comparable from station to station, seasonal changes and the different maximal solar zenith angles account for the differences in total yearly doses. Ratioing between UV-B and PAR allows the detection of subtle changes in the local light climate, due, for example, to mini-ozone holes encountered in northern Europe during spring. Comparison of satellite ozone data with terrestrial ELDONET measurements revealed an overall weak correlation between these data sets. However, local weather conditions, solar zenith angle and latitude as well as reflectivity (i.e. clouds and aerosol; satellite data) show a much stronger correlation to the doses received. The close relationship between the spectral sensitivity of the UV-B sensor used in the ELDONET dosimeter and the CIE erythemal action spectrum allows determination of the erythemal dose on the basis of the dosimeter readings.

Photosynthesis and photosynthetic pigments in the flagellate Euglena gracilis - as sensitive endpoints for toxicity evaluation of liquid detergents.

The present study was designed to validate the applicability of photosynthetic performance using a PAM fluorometer and photosynthetic pigments in Euglena gracilis as endpoint parameters in toxicity assessment of liquid detergents using a dish washing liquid detergent during short- (0-72h) and long-term (7days) exposure. In short-term experiments, the detergent affected the photosynthetic efficiency with EC50 values (calculated for Fv/Fm) of 22.07%, 7.27%, 1.4% and 2.34%, after 0, 1, 24 and 72h, respectively. The relative electron transport rate (rETR) and quantum yield measured with increasing irradiances were also inhibited by the detergent. The most severe effect of the detergent on the light-harvesting pigments (µgmL(-1)) was observed after 72h where chlorophyll a and total carotenoids were decreased at concentrations above 0.1% and chlorophyll b was decreased at concentrations above 0.5%. In long-term experiments, the detergent reduced the photosynthetic efficiency of cultures giving an EC50 value of 0.867% for Fv/Fm. rETR and quantum yield with increasing irradiance were shown to be adversely affected at concentrations of 0.1% or above. A decrease in chlorophyll a and total carotenoids (µgmL(-1)) was observed at concentrations of 0.05% detergent or above. Chlorophyll b was shown to be comparatively less affected by detergent stress, and a significant decrease was observed at concentrations of 0.5% or above. However, there was no prominent decrease in per cell (Euglena) concentration of any pigment. It can be concluded that photosynthesis and light-harvesting pigments in E. gracilis were sensitive to detergent stress and can be used as sensitive parameters in toxicity assessment of detergents in aquatic environments.

Microbial contamination of drinking water in Pakistan--a review.

Water pollution with pathogenic microorganisms is one of the serious threats to human health, particularly in developing countries. The main objective of this article is to highlight microbial contamination of drinking water, the major factors responsible for microbial contamination, and the resulting health problems in Pakistan. Furthermore, this study will be helpful for researchers and administrative agencies to initiate relevant studies and develop new policies to protect further deterioration of water supply with pathogenic microbes and ensure clean and safe drinking water to the public in Pakistan. In Pakistan, water at the source, in the distribution network, and at the consumer tap is heavily polluted with coliforms and fecal coliforms all over the country. An overview of more than 7,000 water samples reviewed here reveals that an average of over 71 and 58 % samples in the country was contaminated with total coliforms and fecal coliforms, respectively. Drinking water contamination accounts for 20 to 40 % of all diseases in the country, which causes national income losses of Rs 25-58 billion annually (US$0.25-0.58 billion, approximately 0.6-1.44 % of the country's GDP). Improper disposal of industrial and municipal wastes is the most important factor responsible for water pollution in the country followed by cross-contamination due to old and leaking pipes and lack of water filtration and disinfection facilities. There is an urgent need for emergency steps to stop further deterioration of water quality and improve the existing water quality so as to protect the public from widespread waterborne diseases.

Ultraviolet radiation and cyanobacteria.

Cyanobacteria are the dominant photosynthetic prokaryotes from an ecological, economical, or evolutionary perspective, and depend on solar energy to conduct their normal life processes. However, the marked increase in solar ultraviolet radiation (UVR) caused by the continuous depletion of the stratospheric ozone shield has fueled serious concerns about the ecological consequences for all living organisms, including cyanobacteria. UV-B radiation can damage cellular DNA and several physiological and biochemical processes in cyanobacterial cells, either directly, through its interaction with certain biomolecules that absorb in the UV range, or indirectly, with the oxidative stress exerted by reactive oxygen species. However, cyanobacteria have a long history of survival on Earth, and they predate the existence of the present ozone shield. To withstand the detrimental effects of solar UVR, these prokaryotes have evolved several lines of defense and various tolerance mechanisms, including avoidance, antioxidant production, DNA repair, protein resynthesis, programmed cell death, and the synthesis of UV-absorbing/screening compounds, such as mycosporine-like amino acids (MAAs) and scytonemin. This study critically reviews the current information on the effects of UVR on several physiological and biochemical processes of cyanobacteria and the various tolerance mechanisms they have developed. Genomic insights into the biosynthesis of MAAs and scytonemin and recent advances in our understanding of the roles of exopolysaccharides and heat shock proteins in photoprotection are also discussed.