Autonomic behavioral impairment induced by simazine exposure during early life of male mouse is mediated by Lmx1a/Wnt1 pathway.

Simazine is a widely used herbicide and known as an environmental estrogen. Multiple studies have proved simazine can induced the degeneration of dopaminergic neuron resulting in a degenerative disease-like syndrome. Herein, we explored the neurotoxicity of simazine on the dopaminergic nervous system of embryos and weaned offspring during the maternal gestation period or the maternal gestation and lactation periods. We found that simazine disturbed the crucial components expression involved in Lmx1a/Wnt1 pathway of dopaminergic neuron in embryonic and weaned offspring. Furthermore, morphological and behavioral tests performed on weaned male offspring treated by simazine suggested that the grip strength, autonomic exploring, and the space sense ability were weakened, as well as the pathological damage of dopaminergic neuron was clearly observed. But, the same neurotoxicity of simazine is less significantly observed in female offspring. Our findings will provide reliable reference for the determination of environmental limits and new insight into the pathogenesis of nonfamilial neurodegenerative diseases related to environmental risk factors.

Injury to dopaminergic neurons development via the Lmx1a/Wnt1 autoregulatory loop induced by simazine.

Simazine is a kind of persistent organic pollutant that is detected in both ground and water and has several routes of exposure. Here, we explored the mechanisms underlying simazine-related effects on dopaminergic neurons via development-related factors using mouse embryos and embryonic mesencephalic hybrid cell line (MN9D cells). We treated pregnant mice with 50 µg/kg bw, 200 µg/kg bw simazine from the 0.5 day to the 10.5 day of embryonic phase and MN9D cells with 600 µM simazine for 24 h to research the mechanism of dopaminergic neurons acute respond to simazine through preliminary experiments. Protein expressions of LIM homeobox transcription factor 1-alpha (Lmx1a) and LIM homeobox transcription factor 1-beta (Lmx1b) displayed a dose- and time-dependent increase after the exposure to simazine. In the 200 µg/kg bw of embryos and the 24h-600 µM of MN9D cells, protein levels of dopaminergic developmental factors were significantly upregulated, and dopaminergic function was significantly damaged for the abnormal expression of Dyt5b. We demonstrated simazine induced the injury to dopaminergic neurons via the Lmx1a/wingless-related integration site 1 (Wnt1) and Lmx1b pathways. In the transfection experiments, we knocked down Lmx1a and Lmx1b of cells to verify the potential target of simazine-induced injury to dopaminergic neurons, respectively. We detected the protein and mRNA levels of development-related genes of dopaminergic neurons and intracellular dopamine levels in different treatment groups. Based on our experiments' results, we demonstrated an acute response to 24 h-600 µM simazine treatment, the simazine-induced injury to dopaminergic neuronal which leads to abnormal dopamine levels and dopaminergic impairment is via the activation of the Lmx1a/Wnt1 autoregulatory loop. Lmx1a is a promising target in the search for the mechanisms underlying simazine-induced dopaminergic injury.

Effect on the dopaminergic metabolism induced by oral exposure to simazine during the prepubertal period in rats.

The herbicide simazine is widely used in agricultural and non-agricultural fields. Studies have shown that simazine inhibits the proliferation of dopaminergic cells and affects the developmental differentiation of dopamine neurons. However, little is known about the effects of simazine on dopaminergic metabolism. Therefore, the present study examined the effects of simazine on Sprague­Dawley (SD) rats from weaning to puberty (40 days exposure). Simazine was administered orally to SD rats at doses of 0, 12.5, 50 and 200 mg/kg body weight. The contents of dopamine (DA), levodopa, dihydroxy-phenyl-acetic acid and homovanillic acid in the striatum were then examined by high-performance liquid chromatography with a fluorescence detector. Quantitative polymerase chain reaction and western blotting were used to analyze the mRNA and protein expression of aromatic amino acid decarboxylase (AADC), tyrosine hydroxylase, orphan nuclear hormone (Nurr1), dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2), monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT). The results indicated that simazine influenced the synthesis, transport and metabolism of DA and led to a reduction of DA levels in the striatum. One potential underlying mechanism is decreased levels of Nurr1, DAT and VMAT2 impacting upon the transport of DA; another is the decreased level of AADC and increased levels of MAO and COMT impacting upon the synthesis and metabolism of DA. These factors may eventually lead to neurological disorders of the dopaminergic system.

Dopaminergic Dysfunction in Mammalian Dopamine Neurons Induced by Simazine Neurotoxicity.

Many studies have shown that the pollutant simazine (6-chloro-N,N'-diethyl-1,3,5-triazine-2,4-diamine), which has been overused, inhibits the proliferation of mammalian dopaminergic cells, and affects the developmental differentiation of mammalian dopaminergic neurons. However, few studies have shown the effects of simazine on dopaminergic metabolism in these cells. Therefore, we aim to examine the metabolic effects of simazine exposure in mouse dopaminergic progenitor neurons (MN9D) at different exposure times. The cells were treated with simazine at 0, 150, 300 and 600 µM for 12, 24 and 48 h, respectively. The content of dopamine in these cells was then examined using the enzyme-linked immunosorbent assay (ELISA) kit. Real-time quantitative polymerase chain reaction (PCR) and western blotting were performed to analyze the mRNA and protein expression of aromatic amino acid decarboxylase (AADC), tyrosine hydroxylase (DYT5b), dopamine transporter (DAT), monoamine vesicular transporter 2 (VMAT2), monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT). The results showed that simazine influenced the metabolism of dopamine and led to a decrease in dopamine level in these cells which may eventually lead to neurological disorders of the dopaminergic system.

Safety assessment of nanopesticides using the roundworm Caenorhabditis elegans.

The extensive use of pesticides is causing environmental pollution, affecting animal organisms in different habitats and also leading human health at risk. In this study, we present as an alternative the use of nanoparticles loaded with pesticides and report their toxicological assessment to a soil organism, Caenorhabditis elegans. Three nanoparticle formulations were analyzed: solid lipid nanoparticles loaded or not with atrazine and simazine, SLN; polymeric nanoparticles, NC_PCL loaded with atrazine; and chitosan/tripolyphosphate, CS/TPP, loaded or not with paraquat. All formulations, loaded or not with pesticides, increased lethality in a dose- dependent manner with similar LC50. Both loaded and unloaded NC_PCL were the most toxic formulations to developmental rate, significantly reducing worms length, even at low concentrations. In contrast, both CS/TPP nanoparticles were the least toxic, not affecting reproduction and body length at higher concentrations, probably due to the biocompatibility of chitosan. The physico-chemical characterization of nanoparticles after incubation in saline solution (used in exposure of organisms) has shown that these colloidal systems are stable and remain with the same initial characteristics, even in the presence of saline environment. Notably, our results indicate that the observed effects were caused by the nanoparticles per se. These results suggest that the development of nanoparticles aiming agriculture applications needs more studies in order to optimize the composition and then reduce their toxicity to non-target organisms.

Fast pesticide detection inside microfluidic device with integrated optical pH, oxygen sensors and algal fluorescence.

The necessities of developing fast, portable, cheap and easy to handle pesticide detection platforms are getting attention of scientific and industrial communities. Although there are some approaches to develop microchip based pesticide detection platforms, there is no compact microfluidic device for the complementary, fast, cheap, reusable and reliable analysis of different pesticides. In this work, a microfluidic device is developed for in-situ analysis of pesticide concentration detected via metabolism/photosynthesis of Chlamydomonas reinhardtii algal cells (algae) in tap water. Algae are grown in glass based microfluidic chip, which contains integrated optical pH and oxygen sensors in a portable system for on-site detection. In addition, intrinsic algal fluorescence is detected to analyze the pesticide concentration in parallel to pH and oxygen sensors with integrated fluorescence detectors. The response of the algae under the effect of different concentrations of pesticides is evaluated and complementary inhibition effects depending on the pesticide concentration are demonstrated. The three different sensors allow the determination of various pesticide concentrations in the nanomolar concentration range. The miniaturized system provides the fast quantification of pesticides in less than 10min and enables the study of toxic effects of different pesticides on Chlamydomonas reinhardtii green algae. Consequently, the microfluidic device described here provides fast and complementary detection of different pesticides with algae in a novel glass based microfluidic device with integrated optical pH, oxygen sensors and algal fluorescence.

Influence of Three Citrus Herbicides on Potential Production of Sorghum bicolor 'Topper 76-6' as a Bioenergy Crop.

Planting bioenergy crops on land previously used for citrus production may offer an alternative source of revenue for growers looking for alternative-to-citrus crops. However, residual herbicides used in citrus production may adversely affect alternative crops. This study evaluated effects of three herbicides (bromacil, norflurazon, and simazine) commonly used in citrus production on the bioenergy crop Sorghum bicolor 'Topper 76-6'. Plants were exposed to herbicides in soil for 1-5 weeks and observations of effects on photosynthetic quantum yield, leaf greenness, height, and biomass were made. Results indicate that concentrations of bromacil and norflurazon greater than 0.09 and 0.07 mg/kg and simazine >0.46 mg/kg will impair growth and development in similar soils. Concentrations below these may also be toxic.

Continued Studies on the Effects of Simazine on the Liver Histological Structure and Metamorphosis in the Developing Xenopus laevis.

This study continued our previous work (Sai et al. in Bull Environ Contam Toxicol 95:157-163, 2015a) by analysing the effects of simazine on the liver histological structure and metamorphosis in the developing Xenopus laevis. Tadpoles (Nieuwkoop-Faber stage 46) were exposed to simazine at 0.1, 1.2, 11.0 and 100.9 µg/L for 100 days. When tadpoles were exposed to simazine at 11.0 and 100.9 µg/L, an increased mortality and damaged liver tissues were observed together with significant inhibition of percent of X. laevis completing metamorphosis on days 80 and 90 and prolonged time of completing metamorphosis. On the other hand, we found that simazine has no significant effects on liver weight and altered hepatosomatic index. Results of this study may be considered to inform risk assessment of the effects of simazine on the development of X. laevis.

Effects of Simazine Exposure on Neuronal Development-Related Factors in MN9D Cells.

BACKGROUND Simazine is a triazine herbicide used worldwide in both agricultural and non-agricultural fields that is frequently detected in surface water and groundwater. Due to its widespread use, an increasing amount of research has focused on the potentially serious environmental and health risks. MATERIAL AND METHODS We used Western blotting and real-time quantitative PCR to analyze the effects of simazine on dopamine neuronal development-related factors in MN9D dopaminergic cells. RESULTS The expression of tyrosine hydroxylase (TH) mRNA was significantly increased after treatment with 300 and 600 µmol L-1 simazine after 24 and 48 h. Levels of nuclear-related receptor 1 (Nurr1) mRNA after 24- and 48-h exposure were decreased with 50 µmol L-1 simazine, but increased with 600 µmol L-1 simazine. Significant increases in TH and Nurr1 protein were observed in all simazine-treated groups at 24 and 48 h. The expression of neurogenin 2 and LIM homeobox transcription factor 1 beta (Lmx1b) mRNA were significantly increased after exposure to 600 µmol L-1 simazine for 48 h, while the expression of wingless-type MMTV integration site family member 1 (Wnt1) mRNA was increased by all doses of simazine. CONCLUSIONS Simazine may have an impact on TH in MN9D cells through 2 mechanisms; one mechanism is through the Lmx1a/Ngn2 pathway, and the other mechanism is through the Lmx1b-pitx3/Wnt1-Nurr1 pathway. These 2 pathways likely do not operate in isolation, but rather together, during the cellular response to simazine exposure.

How benthic diatoms within natural communities respond to eight common herbicides with different modes of action.

Herbicides are common pollutants of rivers in agricultural regions. These contaminants include various types of chemicals with different modes of toxic action. Herbicides can have toxic effects on freshwater benthic diatoms, the base of the aquatic food web. We examined the effects of (non-mixture) herbicide exposure to the health of diatoms for eight common herbicides with three different modes of action; the photosystem II (PSII) inhibitors: atrazine, simazine, hexazinone, tebuthiuron and diuron; two auxinic herbicides: MCPA and 2,4-D; and the EPSP synthase inhibitor: glyphosate. Benthic diatoms within riverine communities were exposed to each herbicide in rapid toxicity tests at concentrations of 50, 200 and 500µgL(-1). The most sensitive taxa were Gomphonema spp. and Encyonema gracilis. Navicula cryptotenella was the most tolerant to herbicide exposure. There was no significant effect of the different herbicide modes of action at the community level. Herbicide mode of action did not alter which taxa were most sensitive within the community and sensitivity rankings of the dominant diatom taxa were similar for each of the eight herbicides. The consistency of the results between herbicides suggests that freshwater benthic diatoms may be suitable in situ indicators for detecting the toxicity of herbicides with differing modes of action.

The Effects of Simazine, a Chlorotriazine Herbicide, on the Expression of Genes in Developing Male Xenopus laevis.

Simazine was investigated for gene expression concurrent with simazine-induced phenotype changes during development of male Xenopus laevis. X. laevis tadpoles (Nieuwkoop-Faber stage 46) were exposed to 0.1, 1.2, 11.0 and 100.9 µg/L simazine for 100 days. The results showed that an increased mortality of X. laevis, decreased gonad weight and altered gonadosomatic index of males significantly (p<0.05) when exposed to simazine at 11.0 and 100.9 µg/L. Significant degeneration in testicular tissues was observed when tadpoles were exposed to simazine at 100.9 µg/L. To investigate the molecular mechanisms behind the testicular degeneration by simazine, we evaluated gene expression in animals treated with 100.9 µg/L simazine and found that 1,315 genes were significantly altered (454 upregulated, 861 downregulated). Genes involved in the cell cycle control, and amino acid metabolism pathways were significantly downregulated. These results indicate that simazine affects the related gene expressions which may be helpful for the understanding of the reason for the reproductive toxicity of simazine on male X. laevis.

Impaired development of female mouse offspring maternally exposed to simazine.

Simazine is a suspected endocrine disruptor and the second most commonly detected pesticide in surface and groundwater worldwide. We evaluated the toxicity of simazine in female mouse offspring with in utero and lactational exposure to the agent. Pregnant mice were exposed to environmentally relevant doses (from 5 to 500µg/kg) of simazine via oral administration, and their female offspring were then analyzed. The female offspring showed shortened anogenital distance and decreased whole body, ovarian, and uterine weights. Their ovaries showed increased apoptotic granulosa cells. In addition, expression of critical genes involved in regulation of cellular apoptosis and proliferation was significantly downregulated in the ovaries of simazine-exposed mice. Moreover, in vitro exposure of human granulosa cell-derived KGN cells to simazine (0.003-1nM) resulted in decreased viability and proliferation. Thus, the present study demonstrates that maternal exposure to low doses of simazine impairs normal development of female offspring via disturbance of cellular apoptosis and proliferation.

Toxicity endpoint selections for a simazine risk assessment.

BACKGROUND: California uses simazine at one of the highest levels for states in the United States (approximately 2.5 million lbs 2006-2010). Simazine causes neuroendocrine disruption and mammary cancer in test animals. A risk assessment was prioritized by the California Department of Pesticide Regulation because of the nondietary concern for simazine exposure to occupational/nonoccupational simazine users, resident nonusers, and bystanders (especially children and children exhibiting pica) at greatest risk. METHODS: No observed effect levels (NOELs) from animal studies as well as human exposure data were used to determine nondietary values for the above populations. Registrant-submitted and open literature studies focusing on oral (major human route) effects for simazine and the major metabolites desisopropyl-s-atrazine and diaminochlorotriazine were reviewed as part of the hazard identification process. RESULTS: Developmental, reproduction, and chronic studies provided the lowest NOELs for the acute (5 mg/kg/day), subchronic (0.56 mg/kg/day), and chronic (0.52 mg/kg/day) exposure durations, respectively. A benchmark dose (95th percentile) was calculated for mammary tumorigenesis, assuming a threshold mechanism in rats (benchmark dose lower limit [95th percentile; BMDL05 ]: 2.9 mg/kg/day). Margins of exposure and uncertainty factors (100-300×, depending on exposure scenario) were used to characterize risk for designated population subgroups. CONCLUSIONS: Fetal developmental delays, endocrine disruption, and mammary tumors resulted from simazine treatment. Systemic and maternal/fetal effects determined the critical NOELs used in risk assessment. Margins of exposures for most scenarios were below acceptable levels, especially for children who may be bystanders where simazine is applied and children who exhibit pica. This risk characterization raises a concern for long-term effects in humans.

Using transcriptomic profiles in the diatom Phaeodactylum tricornutum to identify and prioritize stressors.

The transcriptomic profile of the marine diatom, Phaeodactylum tricornutum, exposed to several ecologically relevant stressors, was used to develop toxicity identification evaluation (TIE)-like gene expression assays. Algal growth inhibition was measured by flow cytometry to determine exposure concentrations that elicited a sublethal toxic response. P. tricornutum was exposed to concentrations of copper (2 µg L⁻¹), cadmium (5 µg L⁻¹), silver (20 µg L⁻¹), simazine (75 µg L⁻¹), the water accommodated fraction (WAF) of weathered crude oil (5 mg L⁻¹), 50 µg L⁻¹ ammonia, a decreased salinity treatment (15‰), and a mixture exposure of ammonia, decreased salinity and cadmium (10 µg L⁻¹). Analysis of the gene expression via microarray indicated that unique transcriptomic signals were generated for each of the individual treatments. Transcriptomic profiles of ammonia and the mixture treatment overlapped substantially. Photosynthesis related transcripts were altered in the simazine (herbicide) treatment. A transcript involved in degrading hydrocarbons, dioxygenase, had increased abundance after crude oil exposure. Overall, transcriptomic responses in the different treatments were associated with stress responses, membrane transport, transcription and translation and could be linked to contaminant mode of action. The transcriptomic profiles were used to design real-time (quantitative) polymerase chain reaction (qPCR) assays that would link changes in transcript abundance to a particular stressor in a TIE-based approach. At least one transcript for each contaminant tested (copper, cadmium, silver, salinity and ammonia) responded exclusively to that contaminant. With further development of additional transcriptomic markers for each contaminant, this new approach has potential to enhance traditional toxicology bioassays by providing additional lines of evidence to identify biologically relevant stressors within a contaminated ecosystem based on changes in the transcriptomic profile.

Oral exposure to the herbicide simazine induces mouse spleen immunotoxicity and immune cell apoptosis.

The authors investigated the toxic effects of simazine on mice spleen immune cells and the underlying mechanisms. Mice were given simazine at 0, 90, 200, or 400 mg/kg by gastric gavage for 3 weeks. The authors then measured immune cell proliferation and the expressions of apoptosis-related proteins (Bcl-2, Bax, Fas, and caspase-3), spleen cell intracellular [Ca(2+)], cellular oxidative stress level, and immune functions. After 3 weeks, mice exposed to simazine had reduced proliferation of both spleen T and B cells. The number of spleen CD4(+) T lymphocytes decreased with simazine exposure, while CD8(+) T cells remained unchanged. Exposure to simazine resulted in reduced immune function, higher intracellular [Ca(2+)], and oxidative stress. Finally, simazine induced spleen immune cells apoptosis by reducing Bcl-2, while increasing Fas and Caspase-3 level. Overall, the immunotoxicity of simazine may involve the induction of immune cell apoptosis and alterations in the immune and physiological functions of spleen cells.

Effect of chronic exposure to simazine on oxidative stress and antioxidant response in common carp (Cyprinus carpio L.).

We investigated the chronic effect of simazine, an s-triazine herbicide commonly present in aquatic environments, on the antioxidant system and oxidative stress indices in common carp (Cyprinus carpio L.). Fish were exposed to sub-lethal concentrations of 0.06 µg l(-1) (environmental concentration in Czech rivers), 2 mg l(-1), and 4 mg l(-1) for 14, 28 and 60 days. Indices of oxidative stress [reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS)], and antioxidant parameters [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), reduced glutathione (GSH)] in fish brain, gill, muscle, liver, and intestine were measured. Chronic exposure to simazine showed the impact of the increased production of ROS leading to oxidative damage to lipids, proteins, and inhibition of antioxidant capacity. Activity of the antioxidant enzymes SOD, CAT, GPx and GSH in groups with high concentrations (2 mg l(-1), 4 mg l(-1)) increased at 14 and 28 days, but decreased after 60 days exposure (p<0.01) as compared with the control group. Changes in enzyme activity were mainly in the liver, but also in gills and brain. Prolonged exposure to simazine resulted in excess ROS formation finally resulting in oxidative damage to cell lipids and proteins and also inhibited antioxidant capacities in common carp tissue.

Effects of low-concentrations of simazine on early life stages of common carp (Cyprinus carpio L.).

OBJECTIVES: The aim of this study is to assess the toxicity of simazine in different developmental stages of common carp (Cyprinus carpio) on the basis of mortality, early ontogeny, occurrence of morphological anomalies, growth rate, and Fulton's condition factor during and at the conclusion of the test. DESIGN: The toxicity tests were performed on carp according to OECD 210 methodologies. The developmental stages of carp were exposed to simazine at four concentrations, 0.06, (reported concentration in Czech rivers), 60, 600, and 3000 µg/l for 36 days and compared to carp in a non-treated control group. RESULTS: Simazine in concentration 0.06 µg/l had no effect on early life stages of carp. Simazine in concentration 600 and 3000 µg/l caused decrease of mass and total length of carp. Fish exposed to three highest levels of simazine showed alteration of tubular system of caudal kidney. On the basis of histopatological changes the values of LOEC = 60 µg/l, NOEC = 0.06 µg/l for simazine were estimated. CONCLUSIONS: Chronic simazine exposure of early-life stages of common carp affected their growth rate, and histology. Some of the changes were observed only at higher exposures (600, 3000 µg/l), but change founded in caudal kidney was affected in fish exposed to the second lowest concentration tested (i.e., 60 µg/l), which is about 10 µg/l higher than reported in Colorado rivers in recent years. Concentrations of simazine in World rivers have been reported to generally vary in the range 0.0003-49.20 µg/l.

Effects of long-term exposure to simazine in real concentrations on common carp (Cyprinus carpio L.).

The effects of a 90 day simazine exposure at concentrations of 0.06 (reported concentration in Czech rivers), 1, 2, and 4 µg L⁻¹ were assessed in one-year-old common carp (Cyprinus carpio L.). Its influence on biometric parameters, hematology, blood biochemistry, liver biomarkers, and histology was investigated. Biometric parameters of common carp exposed to simazine at 0.06 µg L⁻¹ showed no differences from untreated fish. Simazine concentrations of 1, 2, and 4 µg L⁻¹ caused significant (p<0.01) increase of hepatosomatic indices relative to controls. Hematological profiles showed significant (p<0.01) decrease in leukocyte count relative to controls at all concentrations. Biochemical profiles of common carp exposed to simazine at all concentrations showed significant (p<0.01) increase in activity of alkaline phosphatase. In addition, at concentrations of 1 and 2 µg L⁻¹, there was a significant increase in alanine aminotransferase (p<0.05), and, at 4 µg L⁻¹, a significant increase in total protein (p<0.05), albumins (p<0.05), and alanine aminotransferase (p<0.05) compared with controls. Renal histology revealed severe hyaline degeneration of the epithelial cells of caudal kidney tubules in fish at all exposure levels compared to controls. Chronic exposure of common carp to simazine caused significant shifts in hematological, biochemical, and biometric profiles, and histopathological changes. The results of this study indicate that chronic exposure of simazine has altered multiple physiological indices in fish hematology and biochemistry, which potentially may be a biomarker of simazine toxicity; however, before these parameters are used as special biomarkers for monitoring residual simazine in aquatic environment, more detailed experiments in laboratory need to be performed in the future.

Effects of subchronic exposure to simazine on zebrafish (Danio rerio).

OBJECTIVES: Simazine is a triazine herbicide which has been used for a long period in agriculture and in the aquatic environment for control of weeds and algae. The aim of this study was to investigate the effects of subchronic exposure to simazine on growth and the development of histopathological changes in selected organs (gills, kidney, liver) in Danio rerio. METHODS: Juvenile growth tests were performed on D. rerio according to the OECD method No. 215. Fish at the age of 20 days were exposed to the environmental simazine concentration commonly detected in Czech rivers (0.06 µg.L-1) and a range of sublethal concentrations of simazine (0.6, 6.0 and 60.0 µg.L-1) for 28 days. RESULTS: There were no significant differences (p<0.05) between the specific growth rates (r) of the test groups and those of both control groups. Histopathological examination revealed pathological changes in fish exposed to a simazine concentration of 60.0 µg.L-1. The values of NOEC and LOEC of simazine were 6.0 µg.L-1 and 60.0 µg.L-1. CONCLUSIONS: The environmental concentration of simazine in Czech rivers did not have any effects on the growth and development of histopathological changes in D. rerio.

Growth rate of Pseudokirchneriella subcapitata exposed to herbicides found in surface waters in the Alqueva reservoir (Portugal): a bottom-up approach using binary mixtures.

Previous work showed the existence of ecotoxicity of water samples from the Alqueva reservoir due to the presence of the herbicides atrazine, simazine, terbuthylazine and metolachlor. In the present study we examine the effects of these herbicides singly and as binary mixtures on the growth rate of the microalgae Pseudokirchneriella subcapitata. Usually, the toxicity of mixtures is evaluated in relation to the reference models CA (concentration addiction) and IA (independent action). In this study CA model was selected to evaluate the joint effects of s-triazine herbicides on the growth of algae due to their similar mode of action. Moreover, IA reference model was chosen to evaluate the joint toxicity of the chloroacetanilide metolachlor and the s-triazine herbicides due to their different mode of action. In this study dose ratio was the common deviation obtained on both reference models. In the binary mixtures between atrazine/simazine and atrazine/terbuthylazine the increase of the mixtures toxicity (synergism) was mainly due to atrazine. Also, in the binary mixture between atrazine and metolachlor, atrazine was responsible for the increase (synergism) of the mixture toxicity. In the cases of the binary mixtures between simazine/metolachlor and terbuthylazine/metolachlor, the increase of the mixtures toxicity (synergism) was detected when metolachlor was the herbicide dominant, and antagonism was detected when simazine and terbuthylazine were dominant in both mixtures. This study represents an important step to understand the interactions among herbicides detected previously in the waters of the Alqueva reservoir.