The impact of musk ketone on reproduction in zebrafish (Danio rerio)

Nitro musks are a group of nitrated benzenes mainly used by the fragrance industry as a substitute for natural musk. Two of the most common nitro musks, musk xylene and musk ketone, have been detected in water, fish, human adipose tissue, as well as in human breast milk. In this study, female zebrafish were dietary-exposed to musk ketone, prior to spawning with unexposed males. The fish were exposed for 8 weeks at two different dose levels. Accumulation of the compound in the fish as well as reproductive success were studied. Exposed females had reduced body weight and length, as well as reduced liver- and gonad somatic index. The results from the reproduction study showed a dose-dependent reduction in fecundity. Early life-stage mortality was increased and the median survival time was reduced. In addition, an embryo/larvae toxicity test was performed, using newly fertilized zebrafish eggs from unexposed parental fish. Eggs were exposed to a series of different concentrations of musk ketone via the surrounding water. A NOEC value of 10 μg/l and a LOEC value of 33 μg/l was determined. Conclusively, the present study clearly shows that musk ketone negatively affects reproduction and early life-stage survival in zebrafish.     

Fatal attraction: synthetic musk fragrances compromise multixenobiotic defense systems in mussels

We studied interactions of nitromusk compounds musk ketone and musk xylene and polycyclic musks Galaxolide trade mark (HHCB), Celestolide trade mark (ADBI), Tetralide trade mark (AHTN), and Traseolide trade mark (AITI) with multixenobiotic resistance (mxr) transporters in gill tissue of the marine mussel Mytilus californianus (Conrad, 1837). A competitive substrate transport test with rhodamine B was used to assay modulation of transport activity by musks. All tested musks inhibited the transport activity in the low microm range as indicated by increased accumulation of rhodamine B in the tissue. Compared to known substrates of mxr transporters, the effective concentration range was similar to quinidine and about 100 times higher than verapamil. Musk ketone and musk xylene also inhibited efflux of rhodamine B from gill tissue which was loaded with the dye and subsequently incubated with these compounds. Synthetic musk compounds are persistent environmental pollutants in aquatic environments with a high potential to bioaccumulate. As potent inhibitors of mxr transporters they may also play a role as chemosensitizers that enable toxic mxr substrates to accumulate in cells of aquatic organisms.     

Determining the sensitive developmental stages of intersex induction in medaka (Oryzias latipes) exposed to 17 beta-estradiol or testosterone.

Certain environmentally persistent compounds can adversely affect reproduction by acting as steroid hormone agonists or antagonists. The goal of the present study was to determine the developmental stage most susceptible to exogenous hormone (estradiol and testosterone) exposure using a small teleost model. In the first (pilot study) of two experiments, medaka (Oryzias latipes), at varying developmental stages, were bath-exposed to 5 micrograms/l 17 beta-estradiol for 24 h. At 5 months of age, fecundity, fertility and embryo and larval viability (reproductive success) were investigated in control and exposed groups. Fish at 1, 1.5, 2 and 5.5 months of age were also sampled, processed and examined histologically for gonadal alteration. No significant differences in mortality, gonadal morphology, body weight, sex-ratio or time to maturity were seen between control and exposed fish. At 5 months, however, when exposure groups were compared to controls, significant differences were seen in reproductive success and viability of offspring. A second experiment exposed embryo stage 10, and 1-, 7- and 21-day-old larvae for 6 days to 15 micrograms/l 17 beta-estradiol or 100 micrograms/l testosterone. No significant differences were seen at 5 months in mortality, body weight, or time to sexual maturity. However, sex-ratios were significantly biased toward female in the stage 10, 1- and 7-day post-hatch estradiol exposure groups. No significant changes in sex-ratio were associated with testosterone exposure at any developmental stage. Further, intersex gonads were observed in fish from all groups exposed to 15 micrograms/l estradiol. Only those fish exposed as newly hatched fry or at 1 week post-hatch displayed intersex gonads following 100 micrograms/l testosterone exposure. Data from these experiments show that newly hatched fry are that life stage most sensitive to hormone exposure and the most appropriate to use in determining effects of known endocrine-disrupting compounds.     

Impact of benzyl butyl phthalate on shoaling behavior in Fundulus heteroclitus (mummichog) populations

Fundulus heteroclitus preference for association with familiar conspecifics of similar body length was impacted by benzyl butyl phthalate (BBP); this was found to be a statically significant result with a p < 0.0001. When presented with equally sized shoals consisting of either large or small fish, the majority of unexposed (84%) and acetone exposed control (82%) fish selected the shoal of large fish. A small number of control fish chose either the shoal of small fish (6% and 10%) or the neutral zone (10% and 8%) where they were clear morphological outliers. Fish exposed to 0.1 mg/L BBP exposure daily for four weeks selected the shoal of small fish more often than unexposed or acetone controls (7.5- and 4.5-fold respectively). They also remained in the neutral zone and displayed agitation at levels more than twice that of control. Agitation and shoal choice disruption are quantifiable behavioral responses that support the use of F. heteroclitus as a model for detecting sub-lethal BBP exposure.     

Environmental risk assessment of alkylphenols from offshore produced water on fish reproduction

Concern has been raised over whether environmental release of alkylphenols (AP) in produced water (PW) discharges from the offshore oil industry could impose a risk to the reproduction of fish stocks in the North Sea. An environmental risk assessment (ERA) was performed to determine if environmental exposure to PW APs in North Sea fish populations is likely to be high enough to give effects on reproduction endpoints. The DREAM (Dose related Risk and Effect Assessment Model) software was used in the study and the inputs to the ERA model included PW discharge data, fate information of PW plumes, fish distribution information, as well as uptake and elimination information of PW APs. Toxicodynamic data from effect studies with Atlantic cod (Gadus morhua) exposed to APs were used to establish a conservative environmental risk threshold value for AP concentration in seawater. By using the DREAM software to 1) identify the areas of highest potential risk and 2) integrate fish movement and uptake/elimination rates of APs for the chosen areas we found that the environmental exposure of fish to APs from PW is most likely too low to affect reproduction in wild populations of fish in the North Sea. The implications related to risk management of offshore PW and uncertainties in the risk assessment performed are discussed.     

Effects of acid mine drainage from an abandoned copper mine, Britannia Mines, Howe Sound, British Columbia, Canada, on transplanted blue mussels (Mytilus edulis)

Juvenile mussels (Mytilus edulis) were transplanted to Howe Sound, British Columbia, Canada, along an apparent pollution gradient of acid mine drainage (AMD) from an abandoned copper (Cu) mine. Cages containing 75 mussels each were placed at a total of 15 stations and were exposed to concentrations of dissolved Cu in surface waters ranging from 5 to 1009 micrograms/l for a period of 41 days. Mussels located at stations closer to the source of AMD at the mouth of Britannia Creek bioaccumulated higher concentrations of Cu and zinc (Zn) in their tissues. Mussel growth was adversely affected by Cu tissue concentrations above 20 micrograms/g dry wt., while declines in survival and condition index occurred in mussels that bioaccumulated greater than 40 micrograms/g dry wt. Cu. Tissue Zn concentrations (117-192 micrograms/g dry wt.) were likely not high enough to have a direct impact on mussel health. Reduced survival of transplanted mussels was supported by an absence of natural mussels in contaminated areas. Phytoplankton was also severely reduced in areas contaminated by mine waters. Based on the weight of evidence, AMD from the Britannia mine had a deleterious impact on mussel survival in a zone extending at least 2.1 km to the north and 1.7 km to the south of Britannia Creek on the east shore of Howe Sound.     

嗜水气单胞菌(Aeromonas hydrophila)引起斑马鱼肠道生理损伤和肠道菌群失调

研究分析了嗜水气单胞菌(Aeromonas hydrophila)感染斑马鱼后肠道生理健康和肠道微生物的变化。以斑马鱼为研究对象,刮伤皮下真皮,使用105 CFU/mL浓度的嗜水气单胞菌暴露6 h后转入清水,分别在暴露前、暴露后6 h、12 h、24 h取样。使用鱼特异性酶联免疫吸附试验(ELISA)试剂盒,检测肠道中紧密连接蛋白ZO-2 (TJP2)含量和乙氧基异戊二烯-O-脱乙基酶(EROD)酶活性,结果表明,嗜水气单胞菌暴露引起了斑马鱼肠道生理损伤,表现为肠道TJP2含量、EROD酶活性在暴露后显著下调。采用16S rRNA高通量测序检测肠道微生物及其菌群结构变化,嗜水气单胞菌暴露斑马鱼后致病菌不动杆菌属(Acinetobacter)明显增加,OTUs数量明显下降,Alpha多样性降低,结果说明嗜水气单胞菌引起了斑马鱼肠道菌群失调。肠道中微生物的多样性与改善肠道上皮和黏膜屏障功能紧密相关,该研究对从肠道健康的角度评价嗜水气单胞菌毒性,探索嗜水气单胞菌的致病机制具有重要意义。     

Critical evaluation of an intercalibration exercise undertaken in the framework of the MED POL biomonitoring program.

The results of an intercalibration exercise among the laboratories participating in the MED POL program for monitoring biological effects of pollutants along the Mediterranean coasts are presented. Three established biomarkers, i.e. lysosomal membrane stability, metallothionein concentration and ethoxyresorufin-O-deethylase (EROD) activity, were intercalibrated. The stability of lysosomal membranes in mussels (Mytilus galloprovincialis Lam.) was assessed with a cytochemical method. The four participating laboratories were able to discriminate between control animals (membrane labilization times ranging from 21 to 35 min) and Cu-exposed animals (40 micrograms/l Cu for 3 days) (labilization times ranging from 4.5 to 7.4 min). The metallothionein concentration was evaluated in digestive gland homogenates of control mussels and of animals exposed to 200 micrograms/l Cd for 7 days. The eight participating laboratories were able to discriminate between controls and treated samples using a spectrophotometric method. The EROD activity was evaluated by 11 laboratories. All laboratories were able to discriminate between liver microsomal preparations obtained from control and from benzo-a-pyrene exposed fish (Dicentrarchus labrax), with values ranging from 0.5 to 15.88 pmol/min/mg protein in controls and from 5.41 to 165.13 pmol/min/mg protein in treated animals. Using S9 fractions, it was possible to correctly identify control and treated fish, with a variation similar to that found using microsomal fractions, albeit with an inevitable difference in specific activity. As a corollary, all laboratories involved produced comparable data and were able to identify pollutant-induced stress syndromes in sentinel organisms. Thus, intercalibration enables the use of biomarkers in large biomonitoring programs.     

Effects of naphthalene and phenanthrene on visual and prey capture endpoints during early stages of the dourado Salminus brasiliensis

Naphthalene (NAP) and phenanthrene (PHE) effects on Salminus brasiliensis, a carnivorous freshwater fish, were investigated using behavioral tests. Larval stages of S. brasiliensis were exposed to water concentrations of 0, 0.04 mg/l, 0.20mg/l and 0.50mg/l for naphthalene and 0, 0.01 mg/l, 0.05 mg/l and 0.1mg/l for phenanthrene during two developmental phases. The prey fish Prochilodus lineatus were not exposed. Visual acuity was measured at the end of phase 2 in individual S. brasiliensis, which were also tested at the end of each phase for number of attacks on prey, number of prey captured, prey capture efficiency, and distance swam. Vision was impaired by PHE exposure, as acuity angles increased in exposed fish. At Stage I 2.3+/-1.2 prey were captured with 46% efficiency in controls compared to 0.4+/-0.3 prey captured with 13.4% efficiency in fish exposed to 0.05 mg PHEl(-1), the LOEL for both endpoints. At Stage II 4.0+/-1.1 preys were captured in controls compared to 2.5+/-0.9 preys captured in fish exposed to 0.01 mg PHEl(-1), the LOEL. Stage II control fish captured prey with 70% efficiency compared to 30% efficiency at Stage II fish exposed to 0.05 mg PHEl(-1), the LOEL. Distance swam was not affected by either NAP or PHE exposure. The exposure of larval stages of S. brasiliensis to realistic water concentrations of PHE impairs foraging skills and could affect recruitment of the species.     

Exposure of sticklebacks (Gasterosteus aculeatus) to cadmium sulfide nanoparticles: biological effects and the importance of experimental design.

Effects of nanoparticles on aquatic organisms have been little studied to date and toxicological data are urgently needed for development of regulatory frameworks for these substances. Here, we report the findings of a study exposing sticklebacks to cadmium sulfide (CdS) as bulk material and quantum dots. Fish were exposed for 21 d in a flow through test system to 5, 50 or 500 microg l(-1) CdS nanoparticles (nCdS) coated in thiol terminated methyl polyethylene glycol (MPEG), bulk CdS or MPEG at 500 microg l(-1) (nominal concentrations). With the exception of the highest nCdS exposure, measured concentrations were approximately one order of magnitude below nominal. A single fish from each group (excluding MPEG) was examined using energy dispersive X-ray analysis (EDX) to localise cadmium, however, cadmium could not be detected in whole body sections. Elevated levels of oxidized glutathione were measured in the gills of fish exposed to 50 and 500 microg l(-1) nCdS. Induction of vitellogenin synthesis was not detected in any of the treatment groups. The number of males engaged in nest-building behaviour following exposure to 500 microg l(-1) nCdS was reduced and livers of 4/6 fish in the same treatment displayed hepatocellular nuclear pleomorphism. The results are discussed emphasising the fundamental importance of experimental design and the need to understand the behaviour of nanoparticles in the aqueous phase.     

Chronic toxicity of tributyltin on development and reproduction of the hermaphroditic snail Physa fontinalis: Influence of population density

Tributyltin (TBT) is toxic to aquatic organisms and occurs widely in sediments and surface waters of American and European rivers and lakes. This study investigated TBT effects on development and population growth rate (r) of the common, hermaphroditic European freshwater snail Physa fontinalis. Egg ropes of similar age (1-3 days old) were exposed to a control (solvent only) and nominal concentrations of 0.01, 1.0 and 10 microg TBT l(-1) in triplicate. Hatching and mortality were recorded during 0-40 days of exposure. At day 40, 18 juveniles were randomly selected from each concentration (i.e., six from each test vessel) and individually exposed to the same concentration of TBT in 50 ml beakers. A cohort of 20 juveniles was allowed to continue developing in the original test vessels, so that individual and grouped results could be compared. Mortality and reproduction were recorded at 48-h intervals throughout the study period (110 days). Abnormal embryonic development was observed at 1 and 10 microg TBT l(-1). Although 50% of eggs hatched at 10 microg TBT l(-1), all these hatchlings failed to survive. Survivorship of hatchlings was significantly reduced by TBT at 1 microgl(-1). In general, there was a delay in egg production in isolated snails when compared with the grouped snails. Survival, fecundity and population growth rate (r) were reduced in both individual and grouped P. fontinalis at 1.0 microg TBT l(-1). Only a decline in r was observed in snails exposed individually to 0.01 microg TBT l(-1).     

Bioconcentration and distribution of 4-tert-octylphenol residues in tissues of the rainbow trout (Oncorhynchus mykiss)

Branched chain alkylphenols are weak oestrogen mimics which are present in the aquatic environment and have been implicated in the feminisation of fish. This study reports the biotransformation, bioconcentration and tissue distribution of the xenoestrogen 4-tert-octylphenol (t-OP) in juvenile rainbow trout. Fish were exposed for 10 days to a concentration of 4 micrograms/l of [14C] t-OP in a flow-through system and were sampled after 1, 4, 7 and 10 days of exposure. t-OP residues were extracted from all tissues and analysed by radio-high-performance liquid chromatography. After 1 day of exposure radioactive residues were detected in all tissues and reached steady state conditions in the whole fish after 4 days of exposure. The concentration of t-OP residues were highest in bile, followed by faeces, pyloric caeca, liver and intestine. In these tissues the majority of alkylphenol was in the form of two metabolites which were identified by gas chromatography-mass spectroscopy as the glucuronide conjugates of t-OP and t-octylcatechol. t-OP accumulated as the parent compound in fat with a bioconcentration factor (BCF) of 1190, and in brain, muscle, skin, bone, gills, and eye with BCFs of between 100 and 260. This study suggests that exposure to water-borne alkylphenols results in rapid conjugation and elimination of the chemical via the liver/bile route, but that high amounts of the parent xenoestrogen can accumulate in a variety of other fish tissues.     

The effect of PCBs on glycogen reserves in the eastern oyster Crassostrea virginica.

Recent declines in Chesapeake Bay oyster populations have been attributed to disease, and reduced water quality from pollution. The stress associated with pollutant exposure may reduce energy available for growth and reproduction. Polychlorinated biphenyls (PCBs) are lipophilic contaminants that may potentially affect mobilization of lipid reserves, increasing reliance on glycogen stores, which could otherwise be utilized to supply energy for gametogenesis. Thus, PCBs may indirectly affect glycogen stores in oysters in a deleterious manner. To test for this effect, reproductively inactive oysters were exposed to PCBs by feeding individuals 0.7 g of algal paste containing 0, 0.35, or 3.5 micrograms PCBs daily for 8 weeks. Additionally, a group of oysters was exposed to PCBs (0, 0.35, and 3.5 micrograms) plus 0.3 g of non-toxic artificial sediment to examine interactive effects of sediment particles and PCBs. Adductor muscle, mantle, and gonadal tissues were analyzed for glycogen content. Results suggest that glycogen content is reduced in the adductor muscle with increasing PCB exposure, but there are no effects of PCBs in the mantle and gonadal tissues.     

Intersexes in Mississippi River shovelnose sturgeon sampled below Saint Louis, Missouri, USA.

The Missouri Department of Conservation detected high organochlorine levels in tissues of fish from the Mississippi River south of Saint Louis. Chlordane, which is both a carcinogen and an endocrine disrupter, reached 2960 micrograms/kg in fillets of shovelnose sturgeon, Scaphirhynchus platyorynchus, and 1926 micrograms/kg in their roe, wet weight basis. Polychlorinated biphenyls (PCBs) and p,p'-dichlorodiphenyldichloroethylene (DDE) in roe have reached 5810 and 780 micrograms/kg, respectively. The purpose of this study was to determine if endocrine disrupters and carcinogens are a potential cause of the significantly diminished reproduction capacity and population declines in the endangered pallid sturgeon, Scaphirhynchus albus. Twenty-four surrogate shovelnose sturgeons were sampled north of Saint Louis where contaminant levels were lower compared with 17 sampled south of Saint Louis where contaminant levels were high. Liver and gonad tissues were formalin-fixed, histologically processed and examined. Neoplasms were not found. The principal finding was that the testes of two of the seven downstream male fish contained ovigerous lamellae, in addition to mature sperm. Since sturgeons are gonochoristic and intersexes are rare and unusual, the occurrence of intersex in 29% of the male Mississippi River shovelnose sturgeon examined from a population contaminated with chlordane and other chemicals raise concern for human health, species biodiversity and preservation of waterway quality. These findings, however, need to be corroborated by expanded studies with a higher sample size.     

In vitro mechanistic differences in benzo[a]pyrene-DNA adduct formation using fish liver and mussel digestive gland microsomal activating systems

Turbot (Scophthalmus maximus) and mussel (Mytilus edulis) microsomes were incubated with DNA to examine if microsomal in vitro metabolism of BaP could result in DNA adducts detected by 32P-postlabelling. Turbot DNA was incubated with benzo[a]pyrene (BaP), NADPH and microsomal activating systems prepared from either livers of unexposed turbot, turbot exposed to BaP or beta-naphthoflavone (beta-NF) or digestive glands from mussels. The beta-NF activating system generated the highest levels of DNA adducts detected in this study (451.7 adducts per 10(8) nucleotides) and were distributed in three discrete adduct TLC spots, one of which (97% of the total adducts) co-migrated with the 32P-postlabelled BaP 7,8-diol, 9,10-epoxide-N2-guanine adduct. Fewer adducts (P < 0.05) were generated by BaP-induced microsomes (9.4-30.6 adducts per 108 nucleotides) but levels were higher (P <0.05) than those generated from untreated fish (3.5 adducts per 10(8) nucleotides). Co-incubation with 500 microM alpha-naphthoflavone (alpha-NF) resulted in 97-99% inhibition in adduct formation implicating cytochrome P450-dependent (CYP) bioactivation however there was some evidence for carry over of BaP in the liver microsomal preparations from BaP injected fish. In contrast to the fish activating systems, no DNA adducts were observed when mussel microsomes were incubated with BaP, DNA and NADPH.     

Sublethal toxicity of orchard stormwater runoff in Sacramento splittail (Pogonichthys macrolepidotus) larvae

The sublethal effects of stormwater runoff from sections of a plum orchard treated with esfenvalerate or diazinon were evaluated in 7-day-old Sacramento splittail (Pogonichthys macrolepidotus) larvae. Fish were exposed to eight runoff samples using the USEPA standard static renewal method for 96 h acute toxicity testing, then transferred to clean water for three-month to assess the survival, growth, histopathological abnormalities, and heat stress proteins (hsp). No significant mortality was observed at 96 h in exposed fish. At one week, histopathological abnormalities included severe glycogen depletion, cytoplasmic protein droplets, vacuolar degeneration, and cell necroses in liver of all exposure groups. Pyknotic nerve cells were seen in brain of one exposure group. Significantly higher cumulative mortality, lower condition factor, and elevated hsp60 and hsp70 levels (p < 0.05) were occurred in several exposure groups. No histopathological abnormalities were observed after three months in any exposure group. This study confirms that standard acute toxicity tests have underestimated the toxicity of stormwater runoff, and although splittail larvae survived the 96 h exposure, they exhibited reduced survival and growth and showed signs of cellular stress even after a three month recovery period.     

Molecular evidence for multiple UDP-glucuronosyltransferase gene familes in fish

The presence of multiple distinct UGT genes in fish was derived by analysis of DNA sequence data derived the zebrafish EST project, confirming indications from previous protein purification studies in another fish species, the plaice, for a diversity of isoforms in lower vertebrates. At least 10 different UGTs can be identified from nucleotide sequence data in zebrafish. Phylogenetic analysis of exon 1 sequences of the zebrafish, plaice and human UGTs indicates that six of these genes are related to the 1A, 1B and 2 families and that a further four genes were of more ancient lineage. Importantly data for the 3' sequences of the zebrafish clones, both from the database and our own sequences of the publicly available clones did not provide any evidence for elaboration of family 1A genes by alternative splicing in this lower vertebrate.     

植物雌激素对鱼类影响的研究进展

植物雌激素是一类植物来源的具有雌激素活性的非甾族杂环多酚类化合物,也是一类潜在的环境内分泌干扰物,可以影响鱼类的生理、生殖等正常活动.本文介绍了植物雌激素的分布与来源,综述了各种植物雌激素对鱼类生长、行为和生殖的影响,并从植物雌激素与受体结合、抑制类固醇类激素的代谢、酶调节等几种可能的途径,讨论了植物雌激素对鱼类内分泌扰乱作用的机理.     

Method for assessing the chronic toxicity of marine and estuarine sediment-associated contaminants using the amphipod Corophium volutator

Acute sediment toxicity tests do not test key life stage events such as moulting and reproduction and therefore do not reveal the longer-term effects of contaminant exposure. A laboratory method is described for determining the chronic toxicity of contaminants associated with whole sediments. The test is conducted using neonates of the estuarine amphipod Corophium volutator at 15 degrees C, salinity 25 psu and a 12 h light:12 h dark photoperiod. The endpoints are survival and growth after 28 days and survival, growth and reproduction of amphipods upon termination of test i.e. reproduction within all control vessels (ca 75 days). The sediment chronic toxicity test was used to investigate the effects of sediments spiked with environmentally relevant preparations of slightly weathered Alaskan North Slope crude oil, including a water-accommodated-fraction (WAF) and a chemically-dispersed (Corexit 9527) WAF. Sediment oil concentrations were quantified using ultra-violet fluorescence. The amphipods exposed to chemically dispersed oil had higher mortality and lower growth rates than control-, Corexit 9527- and WAF-exposed organisms, resulting in reduced reproduction. The described method supplements the standard acute sediment test and would be particularly useful when long-term ecological effects are suspected but acute tests reveal no significant mortality. The sediment chronic test reported herein has shown that sediment that was not evidently toxic during 10-day acute tests could have population-level effects on sediment-dwelling amphipods.     

光照周期对红鳍东方鲀雌鱼繁殖调控的初步研究

光周期是鱼类季节性繁殖最可靠的环境信号。红鳍东方鲀(Takifugu rubripes)是一种春季产卵鱼类, 具有非常高的经济价值。为了探究光周期调控对红鳍东方鲀成熟产卵时间的影响, 在循环水养殖条件下开展了本研究。将90尾亲鱼随机分配至加速组和自然组。自然组亲鱼在3月产卵, 而加速组亲鱼在1月产卵, 最早产卵时间提前约2个月。结果表明, 加速光周期季节变化速率能够促进红鳍东方鲀性腺成熟。光周期调控方法能够应用于红鳍东方鲀全年繁殖调控。