Chironomids: suitable test organisms for risk assessment investigations on the potential endocrine disrupting properties of pesticides

Selecting an appropriate invertebrate assay has been a primary goal of national and international testing programs for endocrine disrupting chemicals. The available information on the endocrine system, its hormones and their modes of action in controlling physiological processes in invertebrates is limited and the selection of appropriate test species still presents a challenge. This paper outlines the development of a higher-tier full life cycle (FLC) test for pesticides with the non-biting midge Chironomus riparius (Insecta, Diptera, Chironomidae). As an insect, C. riparius represents the species’ richest and ecologically one of the most important groups of invertebrates. In addition, the endocrine system of insects is one of the best studied among the invertebrates. Acute and chronic tests with Chironomus spp. are commonly used for testing and risk assessment of agrochemicals. A chironomid FLC test protocol has been developed and its suitability investigated in an inter-laboratory comparison. The protocol used is based on existing OECD and US-EPA test methods. To verify the suitability of the test to generate endpoints that encompass adverse effects on the arthropod endocrine system, a juvenile hormone analog was selected as positive control substance. Results have demonstrated that the proposed chironomid FLC can be performed in separate laboratories and that the selected arthropod juvenile hormone mimic causes effects. However, the observed toxicity is not proof of an endocrine disruptive mechanism and could equally be evoked by other compounds. Contrary to a screening assay, which aims at revealing a substance’s mode-of-action, the FLC test generates robust, population-relevant endpoints that can be used in the risk assessment of agrochemicals. Since the initial results presented in this paper are encouraging we propose to complete the validation of this assay under OECD with high priority.     

Toxicity of Lithium to Three Freshwater Organisms and the Antagonistic Effect of Sodium

Lithium (Li) is the lightest metal and occurs primarily in stable minerals and salts. Concentrations of Li in surface water are typically <0.04mg l^–1 but can be elevated in contaminated streams. Because of the general lack of information concerning the toxicity of Li to common toxicity test organisms, we evaluated the toxicity of Li to Pimephales promelas (fathead minnow), Ceriodaphnia dubia, and a freshwater snail (Elimia clavaeformis). In the laboratory, the concentration of Li that inhibited P. promelas growth or C. dubia reproduction by 25% (IC25) was dependant upon the dilution water. In laboratory control water containing little sodium (2.8mg l^–1), the IC25s were 0.38 and 0.32mg Li l^–1 and in ambient stream water containing 17mg Na l^–1, the IC25s were 1.99 and 3.33, respectively. A Li concentration of 0.15mgl^–1 inhibited the feeding of E. clavaeformis in laboratory tests. Toxicity tests conducted to evaluate the effect of sodium on the toxicity of Li were conducted with fathead minnows and C. dubia. The presence of sodium greatly affected the toxicity of Li. Fathead minnows and Ceriodaphnia, for example, tolerated concentrations of Li as great as 6mg l^–1 when sufficient Na was present. The interaction of Li and Na on the reproduction of Ceriodaphnia was investigated in depth and can be described using an exponential model. The model predicts that C. dubia reproduction would not be affected when animals are exposed to combinations of lithium and sodium with a log ratio of mmol Na to mmol Li equal to at least 1.63. The results of this study indicate that for most natural waters, the presence of sodium is sufficient to prevent Li toxicity. However, in areas of historical disposal or heavy processing or use, an evaluation of Li from a water quality perspective would be warranted.     

Reassessment of ecotoxicities of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane in Japanese quail under the OECD draft new avian one-generation reproduction test guideline

Ecotoxicological hazards of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (p,p'-DDT) were investigated by a one-generation reproduction study using Japanese quail (Coturnix coturnix japonica) under an Organization for Economic Co-operation and Development (OECD) draft new test guideline 206 following acute and subchronic toxicity studies. In the subchronic feeding toxicity study, tremors, convulsions, and deaths were observed with a clear sex difference, males being more susceptible than females. The estimated total number of sperm tended to decrease in a dose-dependent manner at the end of 6-week treatment. In the one-generation reproduction study conducted at dose levels of 0, 6, 30, and 150 ppm, the estimated total number of sperm tended to decrease in a dose-dependent manner with a significant difference at 150 ppm. Tremors were observed in the majority of hatchlings in the 150 ppm group and at lower incidences in the 30 ppm group. Significantly high mortality rate in chicks persisted from treatment week 3-6 in the 150 ppm group and at treatment weeks 4 and 5 in the 30 ppm group. Despite of these severe adverse effects of p,p'-DDT on hatchlings and chicks, fertilization, egg laying, eggshell thickness or embryonic development was hardly impaired by p,p'-DDT or its metabolites. From these results, it appears that the OECD draft new avian one-generation reproduction test guideline is effective for ecological hazard assessment of chemicals.     

炮制对天山假狼毒毒性及化学成分的影响

目的 研究天山假狼毒炮制前后的急性毒性变化及其体内外组分的改变.方法 将小鼠分为生药组1(60.6 g·kg-1)、生药组2(75.75 g·kg-1)、生药组3(90.9 g·kg-1)、生药组4(106.05 g·kg-1)、生药组5(121.1 g·kg-1)、炮制组1(58.6 g·kgq)、炮制组2(73.2...     

Abnormal thymus development and impaired function of the immune system in rats after prenatal exposure to aciclovir

Aciclovir (synonym: acyclovir) causes abnormal thymus development in rats. After treatment on day 10 of gestation a weight reduction of the organ is obvious in 21-day-old fetuses which persists postnatally. Adult male rats exposed in utero to one or three injections of 100 mg aciclovir/kg body wt given to the dam on day 10 of pregnancy showed a reduction of the thymus weight to 333±158 mg and 276±61 mg (control: 428±92 mg;n=10). Corresponding alterations were detectable in female offspring. Liver weight was also decreased and spleen weight (in relation to body wt) was significantly increased in the offspring after the three exposures. In a host resistance model withTrichinella spiralis the function of the immune system of rats prenatally exposed to aciclovir was examined. Six weeks postnatally 10–12 randomly selected male rat offspring of one control and two treatment groups (1 or 3 injections of 100 mg aciclovir/kg body wt on day 10 of gestation) were infected orally with 500Trichinella spiralis muscle larvae. Before and several times after the infection blood was taken from a tail vein or obtained by decapitation for examination of the antibody titers (IgM, IgG, IgA, IgE) to antigens ofT. spiralis. Six weeks after the infection the weight of relevant organs was determined and tongue preparations were used forT. spiralis muscle larvae counting. Aciclovir exposed animals showed a different immune response than control rats. IgM titers in both treatment groups werehigher than in controls two weeks after the infection but not different by the end of the experiment. The IgG and IgE titers in the high dose group werelower than in the other groups at the end of the observation period. IgA antibody titers in the high dose group were alsolower than controls, but only 2 weeks after the infection. The number ofT. spiralis muscle larvae in tongue preparations was higher in the 3×100 mg aciclovir group than in the low dose group or in controls. Our results indicate that morphological thymus alterations and functional deficits of the immune system can be induced by prenatal exposure of rats to aciclovir on day 10 of gestation.     

北青龙衣对大鼠尿液中5种毒性标志物含量的影响

目的 考察北青龙衣鲜品对大鼠尿液中5种毒性代谢物[L-左旋多巴（laevodopa,L-Dopa）、L-酪氨酸（L-tyrosine,L-Tyr）、L-苯丙氨酸（L-phenylalanine,L-Phe）、吲哚乙酸（indoleacetic acid,Int）及尿黑酸（homogentisic acid,HGA）]含量的影响。方法 Wistar大鼠灌胃给予北青龙衣鲜品,周期为1个月,采用MRM方法,建立同时检测5种代谢物的最优方法,并应用Masslynx软件外标法,对各代谢物精准定量。结果 L-Dopa、L-Tyr、L-Phe、Int及HGA的线性范围分别为30~32 000,25~30 000,7.5~8 000,50~10 000,100~10 000 ng·mL^-1,线性良好;与空白组健康大鼠相比,北青龙衣鲜品组尿液中的5种代谢物含量均有所升高。结论 北青龙衣鲜品可影响大鼠体内5种代谢物的正常代谢。     

钩藤总碱的提取工艺和急性毒性及其对人脐静脉内皮细胞的保护作用

目的 利用正交试验优选钩藤最佳提取工艺,研究钩藤总碱对小鼠急性毒性和对过氧化氢(H₂O₂)诱导损伤的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)的保护作用。方法 采用L₉(3⁴)正交试验法,选择乙醇浓度、提取次数、提取时间、物料比为考察因素,用HPLC测定异钩藤碱、钩藤碱、去氢毛钩藤碱和毛钩藤碱含量并计算转移率,以主成分分析综合得分为指标,优选钩藤总碱的最佳提取工艺。选用KM小鼠,灌胃钩藤总碱溶液,进行最大耐受量(maximum tolerated dose,MTD)的测定,观察14 d内小鼠情况变化,后摘眼球取血,测定小鼠血清中的尿素氮(blood urea nitrogen,BUN)、谷丙转氨酶(glutamic-pyruvic transaminase,GPT)、谷草转氨酶(glutamic-oxaloacetic transaminase,GOT)含量变化。测定钩藤总碱对1.0 mmol·L^-1 H₂O₂诱导损伤的HUVEC存活率和上清液乳酸脱氢酶(lactate dehydrogenase,LDH)、内皮素(endothelin-1,ET-1)、一氧化氮(nitric oxide,NO)含量的影响。结果 优选的钩藤总碱最佳提取工艺为用70%乙醇加热回流提取2次,每次2 h,物料比1:8。急性毒性试验表明,给药后14 d内,各组小鼠活动正常、无明显不良反应、无死亡;钩藤总碱溶液对小鼠的MTD为21.0 g·kg^-1·d^-1,相当于临床成人日用量的1 377倍;与空白组比较,钩藤总碱组GPT显著降低(P<0.01)。与空白组比较,模型组HUVEC存活率及上清液中NO含量显著降低(P<0.01或P<0.05),LDH、ET-1含量显著升高(P<0.01);与模型组比较,钩藤总碱5 μg·mL^-1细胞存活率显著升高(P<0.01),LDH、ET-1含量显著降低(P<0.01或P<0.05)。结论 优选的钩藤总碱提取工艺简单可行,钩藤总碱对小鼠急性毒性较小,对HUVEC损伤有保护作用。     

Ecotoxicological assessment of effluents in the Basque country (Northern Spain) by acute and chronic toxicity tests using Daphnia magna straus

Acute pass/fail, multi-concentration tests, and 3-brood chronic toxicity tests with Daphnia magna Straus (Cladocera, Crustacea) were used to characterise industrial and municipal effluents from various sources. The effluents that “passed” the pass/fail tests had 48-h EC₅₀ values >100% and reproduction No Observed Effect Concentration (NOECs) ≥100%, except for one effluent that had a reproduction NOEC of 31.6%. The acute multi-concentration toxicity tests allowed a rapid classification of effluents from Very Toxic (48-h EC₅₀<25%), to Non-Toxic (48-h EC₅₀ >100%). The acute-to-chronic ratio (ACR: 48-h EC₅₀ divided by the NOEC for reproduction) in the studied effluents ranged from 5 to about 100. From these results, we propose a step-wise protocol for assessing effluent toxicity. First, effluent is evaluated by means of simple and rapid pass/fail acute toxicity tests, to discriminate Non-Toxic from potentially Toxic effluents, thus facilitating the establishment of priority actions. Second, 48-h EC _x is estimated to classify effluents on a toxicity scale from Non-Toxic to Very Toxic. Third, chronic multi-concentration tests are used to calculate reproduction NOECs. These parameters combined with data on effluent chemical composition, chemical and hydrological characteristics of receiving waters, and biological quality criteria can be jointly used for more rational regulatory practices and risk assessment of effluents.     

体外全胚胎培养技术及其在药物生殖发育毒性评价中的应用

体外全胚胎培养（whole embryo culture，WEC）技术是一项在受控的体外环境中培养动物胚胎的技术，是研究药物及化学物质在胚胎发育过程中的药理毒理及作用机制的重要方法之一。本文从大鼠和兔的WEC技术发展概况、培养条件、胚胎发育终点的评分系统和拓展的胚胎发育评价终点、药物生殖发育毒理学应用等方面，对WEC技术及近年来研究进展进行介绍。WEC技术是药物生殖发育毒性研究中重要的替代学研究方法之一。其中，对大鼠与兔组合的WEC研究更具有重要价值，可为胚胎发育毒性研究提供更全面的信息。     

蒙药巴特日七味丸不同提取部位体外抗菌活性筛选和急性毒性研究

目的 考察蒙成药巴特日七味丸不同提取部位对临床常见7种致病菌的体外抗菌活性,并对筛选出的有效活性部位进行急性毒性评价。方法 采用微孔板TTC法和微孔板比浊法测定吸光度（OD）值和最低抑菌浓度（minimum inhibitory concentration,MIC）,观察巴特日七味丸不同提取部位的抗菌作用,并初步确定其有效活性部位;以巴特日七味丸水提物为对照,采用小鼠半数致死量（half lethal dose,LD₅₀）和最大耐受量试验对巴特日七味丸有效活性部位进行急性毒性评价。结果 巴特日七味丸6种提取部位,包括石油醚、氯仿、乙酸乙酯、正丁醇、乙酸乙酯-正丁醇和水残余等部位均具有一定的抗菌活性,其中乙酸乙酯-正丁醇部位具有较强的抗菌活性,并对金黄色葡萄球菌和肠炎沙门氏菌的抑菌作用较强,MIC分别为0.04,0.08 mg·mL^-1。巴特日七味丸水提物和有效活性部位均没有出现LD₅₀值;最大耐受量相当于临床成人（体质量70 kg）安全用药剂量的120倍左右,均未出现急性毒性现象。结论 巴特日七味丸不同提取部位均具有抗菌活性,其乙酸乙酯-正丁醇部位活性较强,并且急性毒性较低,其作用机制有待于进一步研究。     

Extrapolation of the laboratory-based OECD earthworm toxicity test to metal-contaminated field sites

The effects of cadmium, copper, lead and zinc on survival, growth, cocoon production and cocoon viability of the earthworm Eisenia fetida (Savigny) were determined in three experiments. In experiment 1, worms were exposed to single metals in standard artificial soil. For experiment 2, worms were maintained in contaminated soils collected from sites at different distances from a smelting works situated at Avonmouth, south-west England. In experiment 3, worms were exposed to mixtures of metals in artificial soil at the same concentrations as those present in the field soils. A survey of earthworm populations was carried out also. Population densities and species diversities of earthworms declined with proximity to the smelting works. No earthworms were found within 1 km of the factory. Comparison of toxicity values for the metals determined in the experiments indicated that zinc is most likely to be limiting earthworm populations in the vicinity of the works. Zinc was at least ten times more toxic to E. fetida in artificial soil than in contaminated soils collected from the field. This difference was probably due to the greater bioavailability of zinc in the artificial soil. The results are discussed in the context of setting protection levels for metals in soils based on laboratory toxicity data.     

Microdialysis as a tool to determine free kidney levels of voriconazole in rodents: a model to study the technique feasibility for a moderately lipophilic drug

Microdialysis has been employed for the in vivo measurement of endogenous compounds and a variety of drugs in different tissues. The applicability of this technique can be limited by drug lipophilicity which can impair the diffusion through dialysis membrane. The objective of this study was to evaluate the feasibility of using microdialysis to study kidney penetration of voriconazole, a moderately lipophilic antifungal triazolic agent (LogD7.4=1.8). Microdialysis probe recoveries were investigated in vitro by dialysis and retrodialysis using four different drug concentrations (0.1-2 microg/mL) at five flow rates (1-5 microL/min). Recoveries were dependent on the method used for the determination as well as on the flow rate, but independent of drug concentration. The average apparent recoveries determined by dialysis and retrodialysis, at flow rate of 2 microL/min, were 21.1+/-1.5% and 28.7+/-2.0%, respectively. Recovery by retrodialysis was bigger than the recovery by dialysis. The average apparent dialysis/retrodialysis recovery ratio in vitro was 0.73 for all concentrations investigated. The differences between retrodialysis and dialysis recoveries were attributed to the drug's binding to the plastic tubing before and after the dialysis membrane which was experimentally evaluated and mathematically modeled. The in vivo apparent recovery determined by retrodialysis in healthy Wistar rats' kidney was 38.5+/-3.5%, similar to that observed in vitro using the same method (28.7+/-2.0%). The in vivo apparent recovery after correcting for plastic tubing binding (25.1+/-2.8%) was successfully used for determining free kidney levels of voriconazole in rats following 40 and 60mg/kg oral dosing. The results confirmed that microdialysis can be used as sampling technique to determine free tissue levels of moderately lipophilic drugs once the contribution of tubing binding and membrane diffusion on the apparent recovery are disentangled.     

2015—2019年连云港市第二人民医院感染真菌患者菌株分布及耐药性分析

目的 分析2015—2019年连云港市第二人民医院真菌培养阳性标本的病原菌分布情况和耐药状况。方法 收集2015年1月—2019年12月在连云港市第二人民医院所有的真菌培养和药敏试验阳性的标本共663例,应用DL-96Ⅱ细菌测定系统的体外诊断试剂DL-96FOUNGUS对样本进行真菌鉴定,以5种常见抗真菌药物为例,采用MIC法开展对样本中已鉴定真菌的药物敏感性试验,并应用WHONET 5.6软件进行对于该试验结果的统计学分析。结果 2015—2019年连云港市第二人民医院共分离出真菌663株。按照菌种类型分类,可得到如下比例：白色假丝酵母菌占49.9%（331/663）、热带假丝酵母菌占15.2%（101/663）、都柏林假丝酵母菌占8.3%（55/663）、光滑假丝酵母菌14.9%（99/663）、其他假丝酵母菌占11.6%（77/663）。白色假丝酵母菌对伏立康唑、氟康唑、伊曲康唑3种抗菌药物仍具有耐药性;热带假丝酵母菌对氟胞嘧啶、两性霉素高度敏感,对伏立康唑、氟康唑、伊曲康唑表现为耐药;光滑假丝酵母菌对氟胞嘧啶和两性霉素B的敏感性最高。结论 白色假丝酵母菌、热带假丝酵母菌、光滑假丝酵母菌是真菌感染的主要常见致病菌,以热带假丝酵母菌为主的多种假丝酵母菌对于氟康唑、伊曲康唑等临床常用抗菌药物耐药性日渐增长。     

Toxicity of cylindrospermopsin, and other apparent metabolites from Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum, to the zebrafish (Danio rerio) embryo

Cyanobacteria produce a diverse array of toxic or otherwise bioactive compounds that pose growing threats to human and environmental health. We utilized the zebrafish (Danio rerio) embryo, as a model of vertebrate development, to investigate the inhibition of development pathways (i.e. developmental toxicity) by the cyanobacterial toxin, cylindrospermopsin (CYN), as well as extracts from various isolates of Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum. CYN was toxic only when injected directly into embryos, but not by direct immersion at doses up to 50 μg/ml. Despite the dose dependency of toxicity observed following injection of CYN, no consistent patterns of developmental defects were observed, suggesting that toxic effects of CYN may not target specific developmental pathways. In contrast, direct immersion of embryos in all of the extracts resulted in both increased mortality and reproducible, consistent, developmental dysfunctions. Interestingly, there was no correlation of developmental toxicity observed for these extracts with the presence of CYN or with previously reported toxicity for these strains. These results suggest that CYN is lethal to zebrafish embryos, but apparently inhibits no specific developmental pathways, whereas other apparent metabolites from C. raciborskii and A. ovalisporum seem to reproducibly inhibit development in the zebrafish model. Continued investigation of these apparent, unknown metabolites is needed.     

基于AQbD理念的吡罗昔康凝胶体外释放研究

目的 基于分析方法质量源于设计（AQbD）理念,建立并优化吡罗昔康凝胶体外释放实验（IVRT）。方法 建立分析目标、确定关键分析属性［体外释放速率（IVRR）、初始采样时间累积释放量（Q₀）、释放度］;基于先前的知识与经验,从分析目标中推导出有影响的方法变量,并利用石川图进行系统总结,对影响的变量进行风险等级评估,筛选出关键方法变量（膜的种类、释放介质的种类、上样量）;对2种上样量（150、300 mg）、3种释放介质（0.9% NaCl溶液、pH 5.5的磷酸盐缓冲液和pH 7.2的磷酸盐缓冲液）和3种膜［混合纤维素膜（MCE）、聚醚砜膜（PES）、聚四氟乙烯膜（PTFE）］进行2×3×3全析因实验设计,采用扩散池法进行IVRT,将各时间点样品进行HPLC定量分析,进一步计算Q₀、释放度和IVRR。利用JMP Pro软件对实验结果进行建模分析,筛选最优参数。参考美国食品药品监督管理局（FDA）、欧洲药品管理局（EMA）要求对建立的IVRT进行膜惰性验证,释放介质验证,线性、精密度和重复性考察,敏感性和特异性考察及耐用性考察。结果 吡罗昔康凝胶 IVRT 采用静态垂直扩散池（扩散面积 1.767 cm²,接收池体积 12 mL）,温度 32 ℃,转速 600 r·min^-1,释放介质为 pH7.2磷酸盐缓冲液、膜为 MCE、上样量为 300 mg,取样时间为 0.5、1.0、2.0、3.0、4.0、5.0、6.0 h,取样方式为全部取样。方法学验证均符合要求。结论 所建立的吡罗昔康凝胶IVRT可靠、耐用、具有区分力。     

2019—2021年河北北方学院附属第一医院浅部真菌病病原菌分布及药敏分析

目的 探讨河北北方学院附属第一医院浅部真菌病病原菌菌种构成、分布特点,以及分离菌株对常见抗真菌药物的敏感性和耐药性。方法 选择2019年4月—2021年12月河北北方学院附属第一医院皮肤科门诊中临床表现及真菌镜检阳性确诊为浅部真菌病的4 026例患者作为研究对象,进行真菌镜检、培养以及菌株鉴定,并进行药敏试验。结果 4 026例浅部真菌病患者中,甲真菌病1 531例,占比38.03%;手足癣1 100例,占比27.32%;体癣661例,占比16.42%;股癣612例,占比15.20%;头癣122例,占比3.03%。浅部真菌病病原菌分布以皮肤癣菌占据主导地位,占比达75.78%,其中以红色毛癣菌、指（趾）间毛癣菌为优势致病菌。酵母菌占比22.18%,其中以白色念珠菌最为多见,占比15.10%。4 026例浅部真菌病以男性多见,男女比为1.39∶1。甲真菌病在男性患者中占比34.36%,在女性患者中占比43.12%,女性显著多于男性（P＜0.05）。体癣、股癣在男性患者中占比分别为18.25%、17.61%,女性分别为13.88%、11.86%,男性显著多于女性（P＜0.05）。4 026例浅部真菌病以21～50岁年龄段多见,头癣则集中在0～10岁。药敏试验结果显示,437株真菌对于6种抗真菌药物的敏感性最高的是特比萘芬,敏感率高达97.25%,其次为克霉唑,敏感率达93.82%。咪康唑、两性霉素、氟康唑、灰黄霉素敏感率依次为78.26%、55.61%、43.25%、33.18%。结论 河北北方学院附属第一医院浅部真菌病以甲真菌病最为多见,红色毛癣菌是最常见的分离菌株,发病人群男性多于女性,21～50岁为高发年龄段,特比萘芬、克霉唑对浅部真菌病各类病原菌均有良好的敏感性。