Identification of potential biomarkers for diazinon exposure to Japanese Medaka (Oryzias latipes) using annealing control primers

A new differential display-polymerase chain reaction (PCR) method based on annealing control primers was used to screen and identify potential biomarkers from differentially expressed genes (DEGs) in medaka exposed to sub-lethal concentration of diazinon (100 ppb). Among the differentially expressed genes identified, the majority were in functional categories of protein biosynthesis, transport and metabolism according to the gene ontology classification. The differential expression of ribosomal protein genes was quantified by real time PCR. The genes encoding ribosomal proteins including L3 and S17 were selected as potential biomarkers for diazinon exposure in medaka fish.     

Identification of potential biomarkers for diazinon exposure to Japanese Medaka (Oryzias latipes) using annealing control primers

A new differential display-polymerase chain reaction (PCR) method based on annealing control primers was used to screen and identify potential biomarkers from differentially expressed genes (DEGs) in medaka exposed to sub-lethal concentration of diazinon (100 ppb). Among the differentially expressed genes identified, the majority were in functional categories of protein biosynthesis, transport and metabolism according to the gene ontology classification. The differential expression of ribosomal protein genes was quantified by real time PCR. The genes encoding ribosomal proteins including L3 and S17 were selected as potential biomarkers for diazinon exposure in medaka fish.     

Immunostimulating effects of Ginkgo biloba extract against toxicity induced by organophosphate pesticide,diazinon in rainbow trout,Oncorhynchus mykiss: innate immunity components and immune-related genes

The immunostimulating and therapeutic properties of Ginkgo biloba (GB) have always been the focus of traditional medicine over thousands of years. During last decade, special attentions were paid to use of GB in aquaculture to enhance fish health and survival. In the present study, we investigated for the first time the immunogenic effects of dietary GB against oxidative and toxicity induced by organophosphate pesticide, diazinon. In non-diazinon-exposed fish, the plasma total immunoglobulin, lysozyme activity, and peroxidase activity significantly elevated after 60-day experiment in fish supplemented with 1 and 2 g GB/kg diet (p < 0.05). The respiratory burst activity and complement activity significantly increased only in groups supplemented with 0.5 g GB/kg diet (p < 0.05). Furthermore, the peroxidase activity, total immunoglobulin, and lysozyme activity significantly declined in groups supplemented with 4 g GB/kg diet during feeding trial (p < 0.05). There were no significant differences in expression of interleukin 1 beta (IL-1β) and transforming growth factor beta 1 (TGF-β1) genes in kidney between control group (non-GB-supplemented fish) and GB-supplemented fish (p > 0.05). In diazinon-exposed fish, all immunity components significantly decreased during exposure in control and those fed 0.5 and 4 g GB/kg diet (p < 0.05). In fish fed 1 and 2 g GB/kg diet, no alternations were found in immunity components during exposure period (p > 0.05). In addition, diazinon induced the expression of IL-1β and TGF-β1 genes in control and fish fed 0.5 and 4 g GB/kg diet (p < 0.05). No significant changes were observed in expression of IL-1β and TGF-β1 genes in fish supplemented with 1 and 2 g GB/kg (p > 0.05). In conclusion, the results of the present study suggest an immunogenic role for dietary GB at optimum dietary levels (1–2 g GB/kg diet) against toxicity induced by diazinon. Nevertheless, GB at high dietary levels (4 g GB/kg diet) showed immunosuppressive effects, which makes it necessary to optimize its levels in diet.

     

Ammonium,Nitrate and Nitrite Nitrogen and Nitrate Reductase Enzyme Activity in Groundnut (Arachis hypogaea L.) Fields After Diazinon,Imidacloprid and Lindane Treatments

Impacts of diazinon (O,O-diethyl O-2-isopropyl-6-methylpyrimidin-4-yl phosphorothioate), imidacloprid [1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine] and lindane (1,2,3,4,5.6-hexachlorocyclohexane) treatments on ammonium, nitrate, and nitrite nitrogen and nitrate reductase enzyme activities were determined in groundnut (Arachis hypogaea L.) field for three consecutive years (1997 to 1999). Diazinon was applied for both seed- and soil-treatments but imidacloprid and lindane were used for seed treatments only at recommended rates. Diazinon residues persisted for 60 days in both the cases. Average half-lives (t_1/2) of diazinon were found 29.3 and 34.8 days respectively in seed and soil treatments. In diazinon seed treatment, NH₄ ⁺, NO₃ ^?, and NO₂ ^? nitrogen and nitrate reductase activity were not affected. Whereas, diazinon soil treatment indicated significant increase in NH₄ ⁺-N in a 1-day sample, which continued until 90 days. Some declines in NO₃ ^?N were found from 15 to 60 days. Along with this decline, significant increases in NO₂ ^?N and nitrate reductase activity were found between 1 and 30 days. Imidacloprid and lindane persisted for 90 and 120 days with average half-lives (t_1/2) of 40.9 and 53.3 days, respectively. Within 90 days, imidacloprid residues lost by 73.17% to 82.49% while such losses for lindane residues were found 78.19% to 79.86 % within 120 days. In imidacloprid seed-treated field, stimulation of NO₃ ^?N and the decline in NH₄ ⁺NO₂ ^?-N and nitrate reductase enzyme activity were observed between 15 to 90 days. However, lindane seed treatment indicated significant increases in NH₄ ⁺-N, NO₂ ^?-N and nitrate reductase activity and some adverse effects on NO₃ ^?N between 15 and 90 days.     

Pesticides residues in the Prochilodus costatus (Valenciennes, 1850) fish caught in the São Francisco River,Brazil

The objective of this study was to determine the levels of pesticides in the fish Prochilodus costatus caught in São Francisco River, one of most important rivers in Brazil. Thirty-six fish were captured in three different areas, and samples of the dorsal muscle and pooled viscera were collected for toxicological analysis. We evaluated the presence of 150 different classes of insecticides, fungicides, herbicides and acaricides by multiresidue analysis technique using liquid chromatography-tandem mass spectrometry (LC-MS/MS), with the limit of detection of 5 ppb. In this study, organophosphorus and carbamate pesticides were detected at the highest levels in the caught fish. Among the 41 organophosphorus pesticides surveyed, nine types were detected (chlorpyrifos, diazinon, dichlorvos, disulfoton, ethion, etrimfos, phosalone, phosmet and pyrazophos) in the muscle, viscera pool, or both in 22 (61.1%) fish. Sampled tissues of 20 (55.6%) fish exhibited at least one of the eight evaluated carbamate pesticides and their metabolites: aldicarb, aldicarb sulfoxide, carbaryl, carbofuran, carbosulfan, furathiocarb, methomyl and propoxur. Fungicides (carbendazim, benalaxyl, kresoxim-methyl, trifloxystrobin, pyraclostrobin and its metabolite BF 500 pyraclostrobin), herbicides (pyridate and fluasifop p-butyl), acaricide (propargite) and pyrethroid (flumethrin) were also detected. In conclusion, P. costatus fish caught in the São Francisco River contained residues of 17 different pesticides, in both muscles and the viscera pool, indicating heavy environmental contamination by pesticides in the study area.     

The suitability of gastropods as biomarkers

The measurement and induction of mixed function oxyigenases (MFOs) of limnetic gastropods were investigated to estimate their suitability as biomarkers. A determination of MFO activities was performed through the measurement of ethoxyresorufin-O-dealkylase (EROD), pentoxy-(PROD) and benzoxyresorufin-O-dealkylase-activity (BROD). Optimal measuring conditions of these activities were investigated in preparations of the digestive gland of 4 species of limnetic gastropods. Results indicate that MFO-activity inhibiting substances are accumulating in the microsomal pellet, the fraction commonly used for the measurement of MFO-activities. Therefore, the fraction used for induction studies was the postmitochondrial supernatant (PMS). EROD, PROD, and BROD activity ofPlanorbis planorbis andPlanorbis carinatus were measured after a treatment with Aroclor 1254 for 1–17 days. Maximal induction of EROD and PROD were 6 and 10 times the value in the control group ofP. carinatus, respectively induction inP. planorbis was lower. BROD-activity could not been measured inP. planorbis. InP. carinatus, BROD-activity increased to a maximum of 2 times after traetment with Aroclor 1254. The level of induction of EROD-activity is comparable to results described in analog studies with fish.     

Tolerance of an Albino Fish to Ultraviolet-B Radiation

We exposed albino and pigmented medakaOryzias latipes to simulated solar ultraviolet-B (UVB) radiation to determine if albino medaka were less tolerant of UVB radiation than medaka pigmented with melanin. There was no difference in the number of albino and pigmented medaka that died during the exposure period. Spectrophotometric analyses of the outer dorsal skin layers from albino and pigmented medaka indicated that, prior to exposure, both groups of fish had similar amounts of an apparent colorless non-melanin photoprotective substance that appears to protect other fish species from UVB radiation. Our results indicate that albino medaka were as tolerant of UVB radiation as pigmented medaka because they had similar amounts of this photoprotective substance in the outer layers of the skin.     

Organophosphorus pollutants (OPP) in aquatic environment at Damietta Governorate, Egypt: implications for monitoring and biomarker responses

The study was carried out from spring 1999 to spring 2001 to monitor the residue levels of organophosphorus pollutants (OPP) in aquatic environment of the drainage canal surrounding a pesticide factory at Damietta Governorate. Water, sediment, and fish samples were collected at six different seasonal periods. OPPs were analyzed by GLC and confirmed using GC-MS. Chlorpyrifos, chlorpyrifos-methyl, malathion, diazinon, pirimiphos-methyl and profenofos were detected in most samples. Chlorpyrifos was dominant in all water and sediment samples. It was ranged from 24.5 to 303.8 and 0.9 to 303.8 ppb in water and sediment samples, respectively. Diazinon level was slightly similar to chlorpyrifos in fish samples. Data based on the grand total concentration of OPP showed that the most polluted samples were collected either at spring 1999 or autumn 2000. They were 675.5 and 303.8 ppb in water samples and 43.0 and 52.2 ppb in fish collected at spring 1999 and autumn 2000, respectively. The obtained results are in parallel to that found in case of cholinesterase activity where the activity of both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was declined at these seasonal period. The activity levels of AChE and BuChE were found to be 77.18% and 59.67% of control at spring 1999 and 78.62% and 85.80% of control, at autumn 2000, respectively. Thus, AChE and BuChE could be used as biomarkers for tracing and biomonitoring OPP pollution.     

Enhanced visual detection of pesticides using gold nanoparticles

The presence of parts per billion (ppb) levels of chlorpyrifos (O,O-Diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate) and malathion (S-1,2-bis(ethoxycarbonyl) ethyl O,O-dimethyl phosphorodithioate), two common pesticides found in the surface waters of developing countries, have been visually detected using gold nanoparticles. Visual detection of the presence of pesticide is possible when the color change occurring by the adsorption of pesticides on gold nanoparticles is enhanced by sodium sulfate. The method presented here is simple and there is no need of sample preparation or preconcentration. The response occurs within seconds and the color change is very clear. The detection is possible if chlorpyrifos and malathion are present up to a concentration of 20 and 100 ppb, respectively. The method shows great potential for on-site pesticide monitoring. The method is also applicable as a qualitative technique for the performance evaluation of various household water filters, which claim pesticide removal.     

Evaluation of a Daphnia magna renewal life‐cycle test method with diazinon

Abstract

Acute and chronic toxicity tests with diazinon (diethyl 2‐isopropyl‐6‐methyl‐4‐pyrimidinyl phosphorothionate) were conducted on Daphnia magna. The 24‐hr static LC50 was 0.86 μL.L^‐1. The sublethal effects of 0.05, 0.1, 0.5, 0.75 and 1.0 ngL^‐1 of diazinon concentrations on the survival, reproduction and growth of D. magna were monitored for 21 days. The algae Nannochloris oculata (5 x 10⁵ cellsmL^‐1) was used to feed the daphnids. The parameters used to determined the effect of the pesticide on D. magna were: mean total young per female; mean brood size; days to first brood; intrinsic rate of natural increase (r); growth; and survival. Reproduction as well as survival was significantly reduced at diazinon concentrations of 0.10 ngL^‐1 and higher. The intrinsic rate of natural increase (r) decreased with increasing concentrations of diazinon. Growth, as measured by body length, was depressed significantly at 0.05 ngL^‐1 of diazinon and higher concentrations. The maximum acceptable toxicant concentration (MATC) was calculated. The chronic data was used to formulate an acute/chronic ratio.     

In ovo nanoinjection of nonylphenol affects embryonic development of a transgenic see-through medaka (Oryzias latipes), olvas-GFP/STII-YI strain

We performed in ovo nanoinjection of 4-nonylphenol (NP) into embryos of a transgenic see-through medaka (Oryzias latipes), olvas-GFP/STII-YI strain, which has two genotypic sex markers, and examined the effects on development and sexual differentiation. The transgene consisted of a green fluorescent protein (GFP) gene fused to the regulatory region of the medaka vasa gene. Germ cell-specific GFP expression was visualized in the gonad through the transparent body wall of the living fish. The development of each embryo was observed after nanoinjection of 2.0, 10, 50, 125, or 250 ng of NP. NP administration caused significant higher mortality at ⩾50 ng egg⁻¹ and inhibited embryonic development, including abnormal hatch and swim-up failure in all treatment groups except 10 ng egg⁻¹ group. However, it did not cause adverse effects on germ cell proliferation by 10 d posthatch (dph) or sex differentiation of survivors by 100 dph. We concluded that single-dose in ovo exposure to nonylphenol affected embryonic development in the medaka but not gonadal development by 10 dph or sexual differentiation in adult fish by 100 dph. Although further investigations might be needed to elucidate the usefulness of nanoinjection of embryos of this strain, present study indicated that the nanoinjection model using olvas-GFP/STII-YI strain medaka has potential for use in evaluating the effects of chemicals on early development and sexual differentiation.     

Diazinon treatment effects on heart and skeletal muscle enzyme activities

Abstract

The effects of low levels of diazinon treatment on four marker enzymes in rat heart and skeletal muscle have been investigated. Adult male Wistar rats were treated twice a week with a dose of 0.5 ml‐kg^‐1day^‐1 diazinon for 28 weeks. Diazinon treated rats gained significantly less weight than Sham‐treated controls. Typical differences in Succinate dehydrogenase (SDH), Lactate dehydrogenase (LDH), Phosphofructo kinase (PFK) and Hexokinase (HK) activities were observed between heart and skeletal muscles. Diazinon feeding had no effect on heart, soleus, gastrocnemius and plantaris SDH, LDH and PFK enzyme activities after 28 weeks. HK activity was significantly increased in sham‐control soieus and plantaris muscles after 28 weeks. Diazinon feeding inhibited HK activity in plantaris muscle after 28 weeks treatment These results demonstrate that chronic low levels of diazinon have little effect on the glycolytic and oxidative activity in heart and skeletal muscle.     

Brief exposure to copper induces apoptosis and alters mediators of olfactory signal transduction in coho salmon

Pacific salmon are particularly susceptible to copper (Cu)-induced olfactory injuries that can ultimately inhibit neurobehaviors critical to survival. However, the molecular mechanisms underlying Cu-mediated olfactory impairment remain poorly understood. In the present study, we conducted a short-term Cu exposure at levels relevant to urban runoff (5, 25 and 50 ppb) , and investigated the roles of impaired olfactory signal transduction and induced apoptosis as underlying mechanisms of olfactory injury. Increased cell death in the olfactory epithelium was evident in coho receiving 4 h exposures to 25 and 50 ppb Cu. Expression of olfactory marker protein (omp), a marker of mature olfactory sensory neurons, also decreased at 50 ppb Cu. Immunohistochemical analysis of coho olfactory epithelium demonstrated a loss of type 3 adenylate cyclase (ACIII) in the apical olfactory epithelium cilia at all levels of Cu exposure, suggesting an inhibitory effect of Cu in olfactory signaling. Accompanying the loss of ACIII in Cu-exposed coho were reduced intracellular cyclic guanosine monophosphate (cGMP) levels in the olfactory rosettes. Collectively, these results support a linkage among the initial steps of olfactory signaling in Cu-induced salmon olfactory injury, and suggesting that monitoring olfactory cGMP levels may aid in the assessment of salmon olfactory injury.     

Spatio-temporal variations of selected pesticide residues in the Kurose River in Higashi-Hiroshima city,Japan

ABSTRACT

This study investigated spatio-temporal variations of selected pesticide residues in the Kurose River in Higashi-Hiroshima city (Hiroshima Prefecture), Japan. Water samples were collected from the river at seven sites every month for 1 year (March 2016 to February 2017). Pesticide residues were extracted from the samples by a solid phase extraction using Sep Pack C₁₈ cartridges. Once extracted, the samples were analyzed for cyanazine, simetryn, fenarimol, isoprothiolane, and diazinon using a reversed-phase high-performance liquid chromatography ultraviolet visible (HPLC-UV Vis) system. The limits of detection were 3.60, 4.10, 2.80, 6.50, and 7.30 ng L^-1 for cyanazine, simetryn, fenarimol, isoprothiolane, and diazinon, respectively. Good recovery rates (88%–102%), and mean percent relative standard deviation range (1.00%–5.70%) (n = 6) were obtained with a spiking at 0.20 µg L^-1. The maximum concentrations of 282, 391, 60, 1086, and 1194 ng L^-1 were obtained for cyanazine, simetryn, isoprothiolane, fenarimol, and diazinon, respectively. Cyanazine was the most frequently detected pesticide (64% of the samples, n = 84), followed by simetryn (58%), and then diazinon (57%). The highest and lowest pesticide concentrations were measured during the periods May–June, and January–February, respectively. Principal component analysis revealed three principal components in which the pesticides were linked to dissolved organic matter and total suspended solids. The major water quality parameters (electrical conductivity, pH, Na⁺, K⁺, Mg²⁺, Ca²⁺, NH₄⁺, NO₃^?, Cl^?, SO₄^2?, NO₂^?, and temperature) showed no clear trends for these pesticides. The presence of simetryn and isoprothiolane was largely attributed to rice paddy farms, whereas diazinon was associated mostly with vegetable farms and orchards. The diazinon and isoprothiolane patterns were consistent with their use of controlling insects and fungi in the prefecture. The maximum diazinon concentration detected was higher than the human safe level specified by the European Union (100 ng L^-1) in Council Directive 98/83/EC. This is of concern because of the bioconcentration potential of these residues in fish and other marine animals consumed by humans.     

Suppression of sexual behavior in male Japanese medaka (Oryzias latipes) exposed to 17beta-estradiol

Mating pairs of Japanese medaka (Oryzias latipes) received 17beta-estradiol (E2; dose, 3 and 30 microg/g body weight daily) in their diet and E2-free diet (control) for two weeks. The effect on reproductive success was assessed in the mating pairs, and sexual and nonsexual behaviors in the male were quantified. The fecundities of the E2-treated groups were reduced significantly (p < 0.05) compared with those of control, and the sexual behaviors (following, dancing, floating, and crossing) of male fish in response to prostaglandin F2alpha (PG)-injected E2-unexposed females were suppressed dramatically. While the fertility of spawned eggs and the frequencies of the nonsexual behaviors of resting and swimming in a straight line performed by male were unchanged in E2-exposed groups. Therefore, sexual behavior in male may be linked with reproductive success in mating pair of madaka exposed to E2. This assay for sexual behavior using PG may provide a useful tool for assessing the effect of endocrine-disrupting chemicals.     

Effect of ambient-level gas-phase peroxides on foliar injury, growth, and net photosynthesis in Japanese radish (Raphanus sativus)

To investigate the effects of ambient-level gas-phase peroxides concurrent with O₃ on foliar injury, photosynthesis, and biomass in herbaceous plants, we exposed Japanese radish (Raphanus sativus) to clean air, 50 ppb O₃, 100 ppb O₃, and 2-3 ppb peroxides + 50 ppb O₃ in outdoor chambers. Compared with exposure to 100 ppb O₃, exposure to 2-3 ppb peroxides + 50 ppb O₃ induced greater damage in foliar injury, net photosynthetic rates and biomass; the pattern of foliar injury and the cause of net photosynthetic rate reduction also differed from those occurring with O₃ exposure alone. These results indicate for the first time that sub-ppb peroxides + 50 ppb O₃ can cause more severe damage to plants than 100 ppb O₃, and that not only O₃, but also peroxides, could be contributing to the herbaceous plant damage and forest decline observed in Japan's air-polluted urban and remote mountains areas.     

Avoidance by olfaction in a fish, medaka (Oryzias latipes), to aquatic contaminants

Behavioral tests using a fish, medaka (Oryzias latipes), from which the olfactory organs were resected confirmed that fish avoid aquatic contaminants such as surfactants and pesticides mainly by olfaction. Control medaka (non-resected) exhibited significant avoidance to 10, 20 and 30 microg liter(-1) of LAS (sodium linear laurylbenzene sulfonate), 90 and 100 microg liter(-1) of fenitrothion (dimethyl 4-nitro-m-toryl phosphorothionate), and 500 microg liter(-1) of POE-ether (polyoxyethylene lauryl ether). By contrast, medaka with bilateral nose resections (BNRM) exhibited no avoidance to 10-50 microg liter(-1) of LAS, 90 and 100 microg liter(-1) of fenitrothion, and 500 microg liter(-1) of POE-ether. Although medaka with unilateral nose resection (UNRM) avoided some concentrations of these toxicants, the UNRM exhibited no avoidance to 10 and 40 microg liter(-1) of LAS and 90 microg liter(-1) of fenitrothion. That is, the avoidance response of the UNRM was less distinct than that of the controls. This difference may have been caused by the deterioration of detecting ability and the paralyzation or adaptation of olfaction.     

Occurrence and dietary exposure assessment of PFOS and PFOA in cultured Trachinotus ovatus in China

In this study, investigation was conducted into concentrations of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in Chinese farmed Trachinotus ovatus between 2014 and 2015 using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) and ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method. The tissue distribution (muscle, skin, liver, kidney and gill) in Trachinotus ovatus was also assessed. The detection frequencies of PFOS and PFOA in fish were 92% and 3%, respectively, and the mean concentrations were 0.392 and 0.015 μg/kg wet weight. The analysis of PFOS distribution in different tissues in Trachinotus ovatus showed the following trend: skin> gill> kidney> liver> flesh. Results revealeded farmed Trachinotus ovatus in China to generally be contaminated with PFOS. Moreover, the average daily intake for Chinese urban residents calculated on the basis of pollution content was 0.268 ng/kg body weight/d (PFOS) and 0.014 ng/kg body weight /d (PFOA), respectively. Both hazard ratio values were less than 1, indicating that exposure levels of PFOS and PFOA through Trachinotus ovatus consumption may not lead to adverse health effects in the Chinese population.     

Summer air quality over an artificial lake

In the summer of 1998, the air quality (indicators: CO, NO, NO₂, O₃) above the water surface of the Lake Balderey (Essen, Ruhr area, North Rhine-Westphalia, Germany), an artificial lake used for recreation purposes, was measured using the Fourier transform infrared spectroscopy (FTIR) and differential optical absorption spectroscopy (DOAS) remote measurement methods. The lake, with an area of 3 km² was created by damming the Ruhr and is surrounded by higher ground. In calm, bright weather conditions, this location results in a low-exchange situation (formation of temperature inversions, cold air dynamics) with a sustained impact on pollutant concentrations over the lake. The results of trace substance measurements (1/2 h mean values) were compared with values from comparison stations (suburban, high traffic and forest) located outside the area of the lake. In general, it was found that mean CO and NO concentrations over the lake were very low (0.3 ppm and 7.5 ppb, respectively). NO₂ values (15 ppb) were some 3.5 times higher than those recorded at the forest station and O₃ values, at 27 ppb, almost reached the same level as at the forest station (30 ppb). Mass flow densities as a function of wind direction, diurnal courses, differences between weekdays and weekends and comparisons with air quality standards are presented for the lake station.