Leaf surface compound fromBrassica oleracea (Cruciferae) induces oviposition byPieris brassicae (Lepidoptera: Pieridae)

Summary Chemicals present on the surface of cabbage (Brassica oleracea L.) leaves were extracted by dipping these leaves for 3 s in dichloromethane followed by a 3 s dip in methanol. When offered in dual choice bioassays using green paper cards as a substrate, the methanol extract stimulated oviposition activity byPieris brassicae L. (Lepidoptera: Pieridae) females. The oviposition stimulant was isolated using medium pressure liquid chromatography, reversed-phase HPLC, ion-pair HPLC and ion exchange chromatography. Using¹H-NMR spectroscopy, the stimulant could be identified as glucobrassicin (3-indolyl-methyl-glucosinolate). When pure glucobrassicin was offered at a dose identical to that in the crude methanol extract, butterflies did not discriminate between these two substrates in a dual choice test. It is argued that a high sensitivity for indole glucosinolates as host recognition factors may confer an adaptive value for these specialist crucifer feeders. The nutritional significance of their precursor tryptophan and the non-volatile nature of the aglycones formed upon enzymic hydrolysis in damaged tissues are proposed as properties of indole glucosinolates that contribute to this possible adaptive advantage.     

Isolation of novel metal-binding proteins distinct from metallothionein from spotted seatrout (Cynoscion nebulosus) and Atlantic croaker (Micropogonias undulatus) ovaries

Metal-binding proteins were isolated from ovaries of the spotted seatroutCynoscion nebulosus and the Atlantic croakerMicropogonias undulatus collected in 1988 near Port Aransas, Texas, USA. Gel-filtration analysis of spotted seatrout trout ovarian cytosolic fraction on Sephadex G-75 revealed the presence of three zincbinding protein fractions. A major zinc/calcium-binding protein fraction had a low molecular weight (M _r)(6 000 to 10 000), similar to mammalian hepatic metallothionein (MT). All the metals were displaced from this fraction following saturation with exogenous cadmium. After exposure of Atlantic croaker to 2 mg cadmium l^–1 seawater for 2 mo, the majority of the cadmium in the ovarian cytosolic fraction was associated with a similar low molecular weight protein fraction. These proteins were further purified by heat treatment and sequential acetone precipitation. Three isoforms were isolated by reversephase high-performance liquid chromatography. All the isoforms were found to be distinct from mammalian MT, based on amino acid composition. The major isoform contained low amounts of cysteine (approximately 5 residues per molecule) and aromatic amino acids, compared to high amounts of cysteine (typically 17 to 20 residues/molecule) and a lack of aromatic amino acids for mammalian MT. All the ovarian protein isoforms contained more glutamate than mammalian MT. The spotted seatrout and Atlantic croaker ovarian isoforms showed a high degree of homology with metal-binding proteins isolated from mammalian gonadal tissues. The results suggest a physiological role for these metal-binding proteins in developing vertebrate ovaries as well as an involvement in the sequestration of cadmium following environmental exposure.     

Effect of Cd on accumulation and loss of 210Po in the mussel Mytilus edulis

As a result of ²¹⁰Po's previously identified association with sulphur-rich proteins, metallothioneins could have a significant effect on the behaviour and fate of ²¹⁰Po in molluscs. Starved control and cadmium-exposed mussels, Mytilus edulis, were fed ²¹⁰Po-labelled algae (Isochrysis galbana) for 5 d and then allowed to depurate in clean sea water. Cadmium-exposed M. edulis accumulated less ²¹⁰Po in the digestive gland and the remainder of the tissue than control mussels, although this was due to a decrease in tissue weight. More than 40% of ²¹⁰Po was identified as being associated with high molecular weight and heat-treated cytosol proteins in M. edulis. Mussels in a starved state are known to recycle as much as 90% of their amino acids. It is proposed that ²¹⁰Po associated with these and other proteins is recycled, explaining why no significant loss of ²¹⁰Po was observed from the remainder of the tissue in either control or Cd-exposed mussels. Cadmium-induced metallothioneins had no effect on the distribution of ²¹⁰Po in M. edulis; <5% associated with the cytosolic fraction was considered to principally contain metallothioneins. It is suggested that ²¹⁰Po's apparent relationship with metallothioneins is coincidental rather than connected with its role in the regulation of metals. Received: 30 March 1998 / Accepted: 3 August 1998     

Voltammetric analysis of metallothioneins and copper (II) in fish for water biomonitoring studies

An major research area in environmental chemistry is the development of methods for the analysis of biomarkers. Metallothioneins are used as biomarkers in studies of heavy metals exposure in water, because metallothioneins are synthesized and accumulated when organisms are exposed to toxic concentrations of pollutants. In this work, simple and sensitive voltammetric methods were developed for metallothionein and copper (II) determinations in fish liver Lepomis gibbosus. Both analytical methodologies were optimized and applied to samples extracted from individuals previously submitted to sub-lethal toxicological trials with copper sulphate (CuSO₄) and cadmium chloride (CdCl₂). The obtained results showed that both methods are very precise, sensitive, and involve simple sample preparation processes. Moreover, metallothioneins showed better correlation with the toxic exposure than Cu²⁺. To the best of our knowledge, this is the first time that hepatic metallothioneins and Cu²⁺ contents are voltammetrically determined in order to be compared in their function as heavy metal biological indicators.     

Isolation and characterisation of zinc-binding proteins distinct from metallothionein from the eggs of the sea urchin Strongylocentrotus intermedius

Two low-molecular-weight zinc-binding proteins were purified from eggs of the sea urchin Strongylocentrotus intermedius (Echinodermata: Echinoidea) by gel-permeation and anion-exchange chromatography followed by high performance liquid chromatography. The characteristics of these proteins are distinct from those of metallothionein. Amino acid compositions show a low or intermediate content of cysteine and high amounts of acidic amino acids, glycine and histidine; they also contain methionine and aromatic residues. On the basis of these characteristics, the S. intermedius zinc-binding proteins appear similar to other metalloproteins isolated from both vertebrates and invertebrates. No typical metallothionein was detected in the extracts from S. intermedius eggs.     

Methylmercury, total mercury and total selenium in four common freshwater fish species from Ya-Er Lake, China

Snakehead fish (Ophiocephalus argus cantor), silver carp (Hypophthalmichthys molitrtix), crucian carp (Carassius carassius), and common carp (Cyprinus carpio) are four common freshwater fish species in China. In this study, the level of methylmercury (MeHg), total mercury (T-Hg), and total selenium (T-Se) in muscle samples of these four fish species from Ya-Er Lake, China, were analyzed using atomic fluorescence spectrometry coupled with high-performance liquid chromatography, and inductively coupled plasma mass spectrometry. The concentrations of MeHg in all the fish species were significantly correlated with those of T-Hg. Higher T-Hg and MeHg concentrations had accumulated in the snakehead fish, which is a strongly predatory fish, than in the other three species. The concentration ratios of MeHg and T-Hg in the muscles of these four fish species were almost equal. Conversely, there was negative correlation between the concentrations of T-Hg and T-Se, which implies that there is a competition between these two elements with respect to bioaccumulation. It is noteworthy that of all the muscle samples tested, the level of T-Hg exceeded the maximum allowable limit in fish [0.4 mg kg⁻¹ (w/w) recommended by the World Health Organization] in 38.46% of those of the silver carp, 18.18% of those of the crucian carp, and 100% of those of snakehead fish. These results show that the consumption of contaminated fish is a potential threat to human health and that necessary preventive measures to safeguard public health should be emphasized.     

Metallothionein induction inMytilus edulis exposed to cadmium

The exposure of mussels,Mytilus edulis, collected from Whitsand Bay, southwest England, in August 1988, to sublethal concentrations of cadmium (400µg l^–1) for 65 d resulted in the induction of metallothionein (MT) synthesis in the soft tissues. In cadmium-exposed mussels, metallothionein concentrations, measured by differential pulse polarography, increased by a factor of three, from 2 to 3 mg g^–1 to a maximum of 9 mg g^–1 after 30 d. No significant changes could be detected in controls. Cadmium accumulated in the soft tissues of mussels correlated significantly with metallothionein concentrations and can be described by the relationship: MT (mg g^–1)=0.045 Cd (µg g^–1)+3.03 (r=0.803,P<0.001). Gel chromatography of heat-treated cytosolic extracts showed that the accumulated cadmium is bound principally to the newly formed metallothioneins. Copper and zinc were also analysed in the whole soft-tissues and in subcellular fractions of cadmium-exposed mussels. Although copper concentrations were not affected by cadmium-exposure, zinc levels were significantly reduced. The results demonstrate that the induction of metallothioneins inM. edulis is a quantifiable biological response to sublethal levels of cadmium exposure.     

The determination of partition coefficients of organic compounds in technical products and waste waters for the estimation of their bioaccumulation potential using reversed phase thin layer chromatography

Reversed phase thin layer chromatography (RPTLC) has been investigated for the estimation of octanol/water partition coefficients (P), an important parameter for the prediction of the environmental behaviour of organic chemicals. A strong correlation between P derived from the traditional octanol/water system and RPTLC has been established over five orders of magnitude. RPTLC data are likewise correlated to results obtained by high performance chromatography (HPLC). Due to the low costs, simplicity and separation power RPTLC is especially suited for the investigation and screening of mixtures of compounds before more complicated tests are involved.

Results from a round robin test on the determination of partition coefficients by HPLC, RPTLC and the batch method indicate the simplicity and accuracy of the RPTLC technique. Applications of the RPTLC‐technique on technical products and industrial waste waters are described.     

氯霉素对大型溞的急性和慢性毒性效应研究

氯霉素是一种具有广谱杀菌作用的抗生素,曾在水产养殖中广泛使用,虽然目前已被列入我国渔药禁用清单,但在水环境中仍被大量检出。为探究氯霉素对水生生物的毒性作用,选择大型溞(Daphnia magna)作为受试生物,研究氯霉素对其急性毒性和慢性毒性效应,同时建立了氯霉素的高效液相色谱(HPLC)分析方法,通过实测浓度分析确保实验过程中氯霉素浓度保持在可接受范围内。结果表明:氯霉素对大型溞的48 h半数抑制浓度(EC50)为129.5 mg·L~(-1),95%置信区间为124.4!150.9mg·L~(-1),对溞类的急性毒性为低毒;长期暴露能抑制大型溞的产溞数量,以繁殖量为毒性指标,21 d无可观察效应浓度(NOEC)为1.25 mg·L~(-1),最低可观测效应浓度(LOEC)为2.50 mg·L~(-1);各暴露组实测浓度范围在配制浓度的80%～110%,保证了实验的有效性。同时,利用实验获得的急慢性毒性数据,计算氯霉素对大型溞的急慢性毒性比(ACR),发现利用慢性毒性求得的ACR值比利用急性毒性EC10求得的ACR值更接近推荐值。研究表明氯霉素对大型溞的急性毒性低,但具有慢性毒性效应,其环境风险不容忽视。     

Different phagostimulants in potato foliage for Manduca sexta and Leptinotarsa decemlineata

Summary. In the past decades, several studies have focused on the identification of feeding stimulants for specialists that feed on solanaceous plants, especially potato (Solanum tuberosum). In the 1950's, a phagostimulant was isolated from potato and tomato for Manduca sexta and characterised as a glycoside. It was suggested that the phagostimulant for M. sexta and Leptinotarsa decemlineata is identical. We tested whether these insects indeed share the same recognition factor. Previous bioassays for L. decemlineata larvae and adults were found to be unsatisfactory, so a new assay system was developed, using starch and wheat flour-based wafers as a neutral substrate. An aqueous extract of potato foliage that was highly active as a stimulant was fractionated by reverse phase medium pressure liquid chromatography (MPLC). Both insects were stimulated by a MPLC fraction that eluted with 25% methanol in water. Further separation of this and the following fraction (35% methanol, stimulatory for the beetle only) by semi-preparative high performance liquid chromatography (HPLC), using gradients of water and acetonitrile, resulted in the isolation of two distinct fractions that stimulated feeding by L. decemlineata. None of the HPLC fractions was active for M. sexta. However, fractions of the flash chromatography with less polarity (45–75% methanol) had a stimulatory effect only on M. sexta. Thus, the two insects do not use a common feeding stimulant, and for both at least two compounds of different polarity are active. Received 3 July 2000; accepted 24 October 2000     

Determination of 4-nonylphenol in carp blood samples by solid phase extraction and chromatographic techniques

In this study, selective methods were developed for isolation, purification, separation and determination of 4-nonylphenol (4NP) in biological samples. Several methods such as high-performance liquid chromatography (HPLC) with fluorescence and diode array detection and gas chromatography coupled to mass spectromotry (GC-MS) are described for the simultaneous determination of 4NP. The nonylphenols in the plasma of fish were extracted through solid phase process by using octadecyl, polymeric and octadecyl endcapping sorbents. The average recoveries in plasma samples spiked with 4NP of levels 1?µg?mL^?1 were between 80–95% using added surrogate standards. The aim of this study was to determine whether trace amounts of 4NP in fish plasma samples could be detected by solid phase extraction and chromatographic methods (HPLC, GC-MS). This technique of monitoring the levels of endocrine-disruptors in plasma samples is consistent, reliable as well as inexpensive.     

Transmission distance of chemical cues from coral habitats: implications for marine larval settlement in context of reef degradation

The present study (Ishigaki Island, Japan) explored the distance of transmission of chemical cues emitted by live versus dead coral reefs (Exp. 1: High performance liquid chromatography (HPLC) analyses with water sampling station at 0, 1, and 2 km away from the reef) and the potential attraction of these chemical cues by larval fish, crustaceans, and cephalopods (Exp. 2: choice flume experiment conducted on 54 Chromis viridis larvae, 52 Palaemonidae sp larvae, and 16 Sepia latimanus larvae). In the experiment 1, HPLC analyses highlighted that the live coral reef (and not the dead coral reef) produced different and distinct molecules, and some of these molecules could be transported to a distance of at least 2 km from the reef with a reduction of concentration by 14–17-fold. In the experiment 2, C. viridis, Palaemonidae sp, and S. latimanus larvae were significantly attracted by chemical cues from a live coral reef (sampling station: 0 km), but not from a dead coral reef. However, only C. viridis larvae detected the chemical cues until 1 km away from the live coral reef. Overall, our study showed that chemical cues emitted by a live coral reef were transported farthest away in the ocean (at least 2 km) compared to those from a dead coral reef and that fish larvae could detect these cues until 1 km. These results support the assumption of a larval settlement ineffective in degraded coral reefs, which will assist conservationists and reef managers concerned with maintaining biodiversity on reefs that are becoming increasingly degraded.     

Influence of development and rearing temperature on the distribution,ultrastructure and myosin sub-unit composition of myotomal muscle-fibre types in the plaice Pleuronectes platessa

Eggs of the plaice Pleuronectes platessa L. were incubated at temperatures of 5, 8, 10, 12 and 15°C in March 1990, 1991 and 1992. The myotomes of yolk-sac larvae contain a single superficial layer of small-diameter muscle fibres which stain intensely for succinic dehydrogenase activity, surrounding 390 to 500 weakly staining inner-muscle fibres of larger diameter. Larvae reared at 15°C only survived for a few days and had significantly more inner-muscle fibres of larger average cross-sectional area than those hatching at 5 to 10°C. Myofibrils occupied 61% of the volume of inner-muscle fibres in 15°C larvae compared with 35 and 36% in larvae hatching at 5 and 10°C, respectively (P(0.01). Following metamorphosis, which occurs between 7 and 10 wk, the myotomes retain the single layer of superficial-muscle fibres characteristic of larvae. A thickening of the superficial-muscle layer is first evident in 4 to 5 mo-old laboratory-reared fish of 20 mm total length (TL) and in 0-group fish caught in June and July. On the basis of the histochemical staining reactions for myofibrillar ATPase and succinic dehydrogenase activities, the myotomes of 1-group (104 mm TL) and adult (280 mm TL) plaice were found to contain a minimum of six distinct muscle-fibre types. Two-dimensional gel electrophoresis and peptide mapping were used to investigate changes in myosin subunit composition during development. Myosin from the inner muscle of larvae contains two isoforms of the phosphorylatable light-chain 2 (LC2_L1 and LC2_L2). Following metamorphosis and during the first year, inner-muscle fibres co-express LC2 isoforms characteristic of the superficial fast-muscle fibres of adult plaice (LC2_F1 and LC2_F2) in addition to the larval isoforms. Fast-muscle fibres isolated from deep layers of the myotomes in adult plaice only contain LC2_F2. In contrast, myosin from larval muscle and adult fast muscle contain apparently identical alkali light chains (LC1 and LC3). Peptide maps of myosin heavy chains (MHCs) from 6 wk-old larvae and 10 wk-old fish that had completed metamorphosis are similar, but distinct from those of 1-group plaice. Further changes in white-muscle MHC composition are evident between 1-group fish of 104 mm TL and adults of 280 mm TL.     

Absorption and retention of polydimethylsiloxanes (silicones) in fish: Preliminary experiments†

Hydrophobicity and bioaccumulation potential of linear and cyclic polydimethylsiloxane (PDMS) oligomers were estimated by reversed‐phase liquid chromatography and feeding experiments with guppies (Poecilia reticulata). PDMS concentrations in fish were determined by capillary column gas chromatography and gas chromatography‐mass spectrometry. In contrast to polychlorinated biphenyls (PCBs), only very small amounts of PDMS were retained by the fish after six weeks feeding.     

Formation of metallothioneins in relation to accumulation of cadmium in the common mussel Mytilus edulis

Accumulation rates of Cd have been studied in the common mussel Mytilus edulis L. under different laboratory conditions. Semi-static and through-flow experiments were carried out at Cd concentrations of 200 ppb and 50/100 ppb in the water, respectively. A linear uptake of Cd throughout the experimental periods of 36 or 124 d was found in all experiments. The accumulation rate was 3.10 ppm d^-1 at 50 ppb in the through-flow water experiment, 4.1 ppm d^-1 in starved and 6.6 ppm d^-1 in fed mussels in the semi-static experiments with 200 ppb Cd in the water. The Cd-accumulation rate in various fractions of soft parts decreased in the order: body>mantle>muscles. Elution patterns obtained from gel-filtrations showed an increasing amount of Cd-binding proteins (metallothioneins) when the body burden of Cd increased. The Cd content in the body fraction of mussels transferred to clean water for 42 d after first being exposed to Cd during 124 d decreased from 564 ppm on a dry weight basis to 417 ppm while the fraction of Cd bound to metallothioneins rose from 22 to 78% during the same period.     

Polycyclic aromatic hydrocarbons in medicinal plants from Syria

The levels of 16 US Environmental Protection Agency polycyclic aromatic hydrocarbons (EPA PAHs) in 10 medicinal plants in different used parts of plant (leaves and flowers) have been determined. The analytical method consists of sample preparation by ultrasonic extraction with dichloromethane followed by silica gel clean-up. Subsequently, the analysis was carried out by reversed-phase high-performance liquid chromatography (HPLC) coupled to both ultraviolet and fluorescence detections in series to insure the detection of all 16 EPA PAHs. It was observed that the sum of the 16 PAHs (ΣPAHs) in the investigated medicinal plants ranged from 47 to 890 μg kg^–1 where the highest ΣPAHs was found in Sage plant sample. Light PAHs were dominants in all studied medicinal plants. The sum of eight genotoxic PAHs (ΣPAH8) have shown a better indicator of the degree of contamination with carcinogenic PAHs compared to benzo[a]pyrene in these products.     

Developmental changes in the composition of myofibrillar proteins in the swimming muscles of Atlantic herring,Clupea harengus

Changes in myofibrillar protein composition during development have been investigated in the swimming muscles of the Atlantic herring Clupea harengus L. using a range of electrophoretic techniques. The main muscle-fibre type of larvae, and the fast- and slow-muscle fibres of adult fish were found to contain distinct isoforms of myosin heavy chain (MHC) and myosin light chain 2 (LC2). Larval LC2 was present as a minor component of adult fast-muscle myosin. In contrast, larval and adult fast-muscle myosin appeared to contain identical alkali light chains. Tropomyosin and troponin C were also identical in larval and in adult fast-muscle. All three muscle-fibre types contained unique isoforms of troponin T (TNT) and troponin I (TNI). Larval muscle had multiple isoforms of TNT, some of which may correspond to embryonic forms. It was concluded that although the main muscle-fibre type in larvae shares some myofibrillar proteins with adult fast muscle, it also contains characteristic isoforms of MHC, TNI, TNT and LC2 and therefore represents a distinct fibre type. The particular combination of myofibrillar proteins present at any developmental stage was found to be dependent on the rearing temperature. For example, a higher proportion of embryonic TNT isoforms were present at hatching in larvae reared at 5°C than at either 10 or 15°C. Over a period of 7 d, there was a gradual reduction in the number of TNT isoforms, but the pattern in 5°C larvae after 7 d still did not resemble that in 1 d-old larvae reared at 15°C.     

Residues of fluoroquinolones in marine aquaculture environment of the Pearl River Delta, South China

Concentrations and distributions of selected fluoroquinolones (norfloxacin, ciprofloxacin and enrofloxacin) in water, sediments and nine kinds of fish species collected from 6 sites in two marine aquaculture regions of the Pearl River Delta, China, were analyzed by using high-performance liquid chromatography with fluorescence detector (HPLC). The results showed that the concentrations of ciprofloxacin and enrofloxacin were below the limits of quantification (LOQ) in all water samples except for norfloxacin. Norfloxacin and ciprofloxacin concentrations ranged from 1.88 to 11.20 ng g⁻¹ dry wt, 0.76–2.42 ng g⁻¹ dry wt in sediments collected from the Dapeng’ao region (sites 1–3) and ranged from 2.31 to 4.75 ng g⁻¹ dry wt, 1.26–1.76 ng g⁻¹ dry wt in sediments collected from the Hailing Island region (sites 4–6), respectively. However, no enrofloxacin was found in all sediment samples. The three fluoroquinolones (FQs) were detected in all fish samples, and the concentrations were higher in liver tissues than those in muscle tissues. The levels of norfloxacin were higher than ciprofloxacin and enrofloxacin in both liver and muscle tissues. Among the nine marine fish species, Siganus fuscescens from Hailing Island had a significantly high level of norfloxacin in liver tissue (254.58 ng g⁻¹ wet wt), followed by Sparus macrocephalus (133.15 ng g⁻¹ wet wt) from Dapeng’ao, and the lowest value was Lutianus argentimaculatus (5.18 ng g⁻¹ wet wt) from Hailing Island. The obtained results of FQs in present study do not represent a risk to the human health in Guangdong coastal area, based on the maximum residue limits (MRLs) established by Chinese Government and the acceptable daily intake (ADI) recommended by the Food and Agriculture Organization and World Health Organization (FAO/WHO).     

Some properties of trypsin-like proteases extracted from the seaweedCodium fragile and their purification

Two enzymes were purified from the green algaCodium fragile, collected in 1988 at Fukuoka, Japan, by batch-wise ion-exchange extraction, affinity chromatography and ion-exchange high-performance liquid chromatography (HPLC) using Boc-Ala-Ala-Pro-Arg-pNA as a substrate. The molecular weights of the enzymes were estimated as 38 000 and 39 000, using gel filtration HPLC. The enzymes had the same optimal pH range of 7 to 9 for both activities, and an exclusively hydrolyzed peptide bond on the carboxyl-terminal side of theL-arginine of peptidep-nitroanilides. The ratios of the enzymatic activity for X-Arg-pNA to X-Lys-pNA were larger than 100. The enzymes exhibited 30 times higher activity toward Boc-Ala-Ala-Pro-Arg-pNA when compared with trypsin. The activities were strongly inhibited by diisopropyl phosphofluoridate (DFP), and partially inhibited by phenylmethylsulfonyl fluoride (PMSF), benzamidine, leupeptin and antipain. The isolated enzymes were presumed to be trypsin-like serine protease from their primary substrate specificities and inactivation behavior.