Effects of aflatoxin B, aflatoxin B, aflatoxin G and sterigmatocystin on viability, rates of development, and body length in two strains of Drosophila melanogaster (Diptera)

Two wild-type laboratory populations of Drosophila melanogaster, Florida-9 (sensitive to aflatoxin (AF) B₁-induced toxicity) and Lausanne-S (resistant to AFB₁-induced toxicity) were tested to determine relative degress of sensitivity to growth from the egg stage on media containing 0.2, 0.6, 2.0, and 4.0 ppm AFB₁, AFG₁, AFB₂, or sterigmatocystin (ST). Data indicate that strain Florida-9 is quite sensitive to AFG₁ toxicity at both the egg-pupa and egg-adult stages of development while Lausanne-S is quite resistant to such toxic effects. For Lausanne-S, AFB₁ > AFG₁ in relative toxicity, while for Florida-9, AFG₁ > AFB₁. The latter is noteworthy since vertebrate studies consistently show that AFB₁ is a significantly stronger carcinogen and mutagen than AFG₁. Possible explanations are discussed. Neither strain tested displayed toxic responses to the presence of AFB₂ or ST in the culture media; however, the 4.0-ppm Lausanne-S treatment displayed a significantly lower adult mortality rate than the control, indicating that Lausanne-S flies may benefit from the presence of ST in the culture medium.     

Toxicological effects of aflatoxin B<Subscript>1</Subscript> on the earthworm <Emphasis Type="Italic">Eisenia fetida</Emphasis> as determined in a contact paper test

In this study, aflatoxin B₁ (AFB₁) toxicity toward the earthworm Eisenia fetida (Savigny 1826) was evaluated in contact paper test systems containing distilled water and ethanol or 20 to 400 μg/ml of AFB₁ over 72 h of exposure. The results indicated that AFB₁ could induce significant damage to earthworms (coiling, curling, excessive mucus secretion, clitellum swelling) at greater than 75 μg/ml. Moreover, AFB₁ had harmful effects on E. fetida (degenerative changes such as bulging of the clitella regions) at levels higher than 150 μg/ml. The calculated LD₅₀ was 168.5 μg/ml. These findings confirm that E. fetida and standardized methods based on this organism (OECD 207 1984) are applicable and useful in mycotoxin related toxicity studies.     

Effects of aflatoxin B1 on the liver and kidney of broiler chickens during development

Aflatoxin B₁ (AFB₁) negatively affects chicken (Gallus domesticus) growth. This effect is more severe during development. We studied the influence of age on the toxic effects of AFB₁ on plasma, renal and hepatic enzymes, under two protocols, in adult and in developing Arbor-Acres chickens. Protocol A: 100 male 4-week-old chickens (640 g), received AFB₁, 0.5, 1.0, or 2.0 μg/g of feed (daily p.o.), a fourth group received an aflatoxin-free diet. Five birds/group were slaughtered at 7, 14, 21 and 28 days of treatment. Body, hepatic and renal weights, succinate-dehydrogenase (SDH) and glutamate-dehydrogenase (GluDH) in plasma and liver were measured. Hepatic SDH and GluDH decreased (P<0.05). Protocol B: two groups of 24 male 1-week-old chickens (106 g) received either aflatoxin-free feed (n=24) or AFB₁ feed (2.0 μg/g). At days 7, 14, 21 and 28, the same parameters of Protocol A were measured. AFB₁ markedly reduced body weight gain (20–30%), plasma proteins, albumin, renal and hepatic protein content (P<0.05) and increased absolute and relative weights of the kidney (P<0.05). SDH and GluDH were reduced (P<0.05), while total renal γ-glutamyltranspeptidase (GGT) increased (P<0.05). Results suggest that serum proteins, SDH and GluDH are sensitive early indicators of this toxicity that was more severe in developing chickens. Decrease in serum albumin might be used as an early and suitable indicator of the deleterious effect of this mycotoxin in developing chickens.     

Pharmacological activity and toxicity of Phencyclidine (PCP) and Phenylcyclohexene (PC), a pyrolysis product

Initial acute behavioral studies in mice indicated that phencyclidine (PCP) produced marked motor impairment as measured by the inverted screen technique with an ED₅₀ value of 4.1 μMole/kg (i.v.). Phenylcyclohexene (PC) was considerably less active with an ED₅₀ value of 325 μMole/kg (i.v.). PCP was also shown to be more lethal than PC as acute (24 hr; i.v. injection) LD₅₀ values (μMoles/kg) in males were 57 and 448, and in females were /6 and 425, respectively. A greater acute lethality was also produced by PCP after i.p. and p.o. administration. Subchronic (14-day) exposure (i.p.) to PCP at doses up to ≈40 percent of the acute LD₅₀ value (123.6 μMole/kg, i.p., daily) was without significant effect on body and organ weights, hematology and clinical chemistry, and humoral and cell-mediated immunity. Higher doses of PCP were not possible because of acute lethality. Subchronic exposure to PC (63.4, 317, and 634.5 μMoles/kg; 4 percent, 20 percent and 40 percent of acute i.p. LD₅₀ value, respectively) produced several marked effects. At the highest dose tested, body weight and thymus weight in both males and females, and liver weight in males were significantly decreased. The spleen weight of males exposed to 317 μMole/kg PC was also significantly decreased. Humoral immunity (production of antibody forming cells) was significantly inhibited in both males and females exposed to PC. In contrast, cell-mediated immunity (development of a delayed-type hypersensitivity response) was only significantly inhibited in females. As PCP has no measurable toxicity under these conditions and PC produced significant effects at relatively high doses, the results suggest that neither chemical is exceptionally toxic following subchronic exposure.     

Mutagenicity and cytotoxicity of the carcinogen-mutagen aflatoxin B1 in Streptococcus pneumoniae (Pneumococcus) and Salmonella typhimurium: dependence on DNA repair functions

Strains R6, R6x and R6uvr-1 of Streptococcus pneumoniae (Pneumococcus) are sensitive to the cytotoxic effects of the mutagen/carcinogen aflatoxin B₁ (AFB₁). R6uvr-1 is more prone to the cytotoxic effects of AFB₁ than the repair-proficient parental strain, R6. The same differential susceptibility of strains R6, R6x and R6uvr-1 was observed when UV light replaced metabolically activated AFB₁. All pneumococcal strains were immutable by AFB₁. AFB₁ mutagenesis in Salmonella typhimurium strains was dependent on a functional RecA gene product. The enhancing effects of ΔuvrB and plasmid pKM101 were found to be additive. Data presented are consistent with the following: (i) AFB₁ toxic effects are due mainly to DNA binding of AFB₁; (ii) AFB₁ mutagenesis is dependent on error-prone DNA repair; (iii) Pneumococcus lacks an active error-prone (SOS) DNA-repair system.     

Quantitative food utilization and reproductive allocation by adult milkweed bugs, Oncopeltus fasciatus

ABSTRACT. Age-specific and lifetime dry mass budgets were estimated for mated and virgin adult milkweed bugs, Oncopeltus fasciatus (Dallas) Hemiptera: Lygaeidae), fed air-dried milkweed seeds ( Asclepias syriacd ) in the laboratory at LD 14:10 and 23°C. Relative consumption rate (RCR) of all bugs was high during the first 8 days posteclosion (teneral period) as their fresh weight, dry weight, and fat content increased. Thereafter, the physiological syndrome associated with reproduction in mated females was indicated by their higher RCR, earlier and greater rate of egg production, greater lifetime relative metabolic rate and higher net and gross production efficiencies than virgin females and males. Males tended to live longer than virgin and mated females, which had similar lifespans. Mated females weighing less at eclosion remained lighter in weight on the day of mean peak weight, but food consumption, egg production and lifespan were independent of body-weight over a 25% range. Input of nymphal reserves or male reproductive secretions to egg production is probably minor in comparison with the adult female's food budget. The high proportion of the food budget allocated to egg production by mated females of O.fasciatus is consistent with its migratory, colonizing lifestyle.     

Effects of mycotoxin pretreatment on aflatoxin B1 post-treatment toxicity in Drosophila melanogaster (Diptera)

Two wild-type laboratory strains of Drosophila melanogaster were used in this study: strain Flordia-9, which is sensitive to aflatoxin B₁ (AFB₁)-induced toxicity, and strain Lausanne-S, which is resistant. Eggs of these strains were deposited on medium containing either low or high doses of dietary AFG₁, AFB₂, or sterigmatocystin (ST) and allowed to develop into second instar larvae. After this pretreatment, the larvae were transferred onto medium containing either high or low doses of dietary AFB₁ (post-treatment) and allowed to complete development and eclose as adults. Viability and development data were analyzed to determine the effects of the various pretreatments on the level of AFB₁-induced toxicity in the post-treatments. In no case did any of the pretreatments reduce the toxic effects of AFB₁ post-treatment responses. However, for strain Florida-9, all high-dose pretreatments resulted in enhanced post-treatment toxicity, and all low-dose pretreatments also enhanced toxicity of high-dose post-treatments. For strain Lausanne-S, high-dose AFB₂ pretreatment significantly enhanced toxicity of both high- and low-dose post-treatments. These results indicate that, in strain Florida-9, pretreatment with relatively less toxic mycotoxins (ST and AFB₂) has an enhancing effect on AFB₁-induced toxicity, whereas in strain Lausanne-S, a similar but smaller enhancing effect is seen only with AFB₂ pretratment.     

Variation in sensitivity to aflatoxin B1 among several strains of Drosophila melanogaster (Diptera).

Eggs of five different wild-type strains of fruit flies, Drosophila melanogaster, were allowed to develop into adults on media containing 0.27 and 0.40 ppm of the toxic and carcinogenic mycotoxin, aflatoxin B₁ (AFB₁). Egg-to-adult viability and development time, adult sex ratio and size, and pupa case size were measured for each treatment and compared to control values for each strain. Adult size and pupa size were not affected significantly by the AFB₁ treatments, while egg-to-adult development time increased for strains grown on AFB₁-containing media. Strain-specific changes in egg-to-adult viability and adult sex ratio were observed. The most probable explanation for these differences is genetic variation among the strains. Crimea, Hikone-R, Lausanne-S, Oregon-R, and Swedish-C were the strains tested.     

Effects of the nematicide oxamyl on life cycle stages of Globodera rostochiensis

Hatched second stage juveniles of Globodera rostochiensis were found to be more sensitive than either unhatched juveniles or adult males to low concentrations of oxamyl (EC₅₀ value for activity 0.5 μg oxamyl ml^-1). Development of juveniles in roots was significantly inhibited by 2.0 μg oxamyl ml^-1 probably due to impairment of normal feeding activity. The behavioural events necessary for orientation of juveniles towards roots for invasion and orientation of males towards females for fertilisation were found to be impaired by 0.5 and 1.0 μg oxamyl ml^-1 respectively. A comparison with previous work on Meloidogyne incognita showed G. rostochiensis juveniles to be more sensitive to oxamyl.     

Inhibition of in vitro aflatoxin B1-DNA binding in rainbow trout by CYP1A inhibitors: α-naphthoflavone, β-naphthoflavone and trout CYP1A1 peptide antibody

Rainbow trout cytochrome P450 (CYP)1A detoxifies aflatoxin B₁ (AFB₁) to aflatoxin M₁ (AFM₁), whereas CYP2K1 activates AFB₁ to AFB₁-8,9-epoxide. We report that α-naphthoflavone (ANF) and β-naphthoflavone (BNF) both strongly inhibit CYP1A-mediated ethoxyresorufin O-deethylase (EROD) activity (K_i = 9.1 ± 0.8 and 7.6 ± 1.1 nM, respectively). These inhibitors (selective for mammalian CYP1A at low concentrations), as well as rabbit polyclonal antibody to a trout CYP1A1 peptide (residues 277–294), also strongly inhibited trout microsome-catalyzed AFB₁-DNA binding and lauric acid (ω-1) hydroxylation in vitro, reactions previously established to be CYP2K1-dependent. ANF at 0.5, 5, 50 and 500 μM inhibited liver microsome-catalyzed AFB₁-DNA binding by 22, 58, 84 and 91%, respectively, whereas BNF at the same concentrations inhibited 22, 74, 78 and 81%, respectively. The CYP1A1 peptide and CYP2K1 polyclonal antibodies (10 mg IgG/mg microsomal protein) inhibited AFB₁-DNA binding by 84 and 66%, respectively, compared with pre-immune IgG. Lauric acid (ω-1) hydroxylation was inhibited 61% by 5 μM ANF, 69% by 5 μM BNF and 100% by either antibody at 12 mg IgG/mg microsomal protein. These results demonstrate that mammalian CYP1A inhibitors also inhibit trout microsomal AFB₁-DNA binding and lauric acid (ω-1) hydroxylation, catalyzed primarily by CYP2K1. In the absence of evidence that trout CYP1A can catalyze AFB₁-DNA binding, the results suggest configuration similarities at, or near, the active sites for these two fish enzymes that result in antibody crossreaction and loss of the inhibitor specificity observed with mammalian CYP1A.     

Quantitative changes and synthesis of cyanoprotein in whole body and tissues during development of the bean bug,Riptortus clavatus

Changes in biliverdin-binding cyanoprotein content in whole body and tissue extracts during development of nymphal and adult (non-diapause) bean bugs, Riptortus clavatus were analyzed by rocket immunoelectrophoresis (RIE). RIE using anti-CPegg serum can be used to determine the content of CP-A (Cp-1, 2 and 3) and CP-B (CP-4) separately. During the nymphal stage CP content of whole body changes cyclically in each instar. In the first nymphal instar, CPegg is the main CP which disappears during the first-second instar ecdysis. In nymphal bugs from the 2nd to 4th instars only CP-B (CP-4) is detected, and at the beginning of each instar the CP content is very low but increases toward the next ecdysis, after which CP decreases and disappears very rapidly. In the 5th nymphal instar, CP-B is the major CP but CP-A (CP-1, 2 and 3) is also detected. These changes in whole body CP content of 5th instar nymphs are observed in both females and males. The content of total CPs in the 5th instar nymph reaches about 1000 μg in the whole insect. During nymphal-adult ecdysis, nymphal CPs decrease and disappear at day 2 after emergence. In female adults CP-A (CP-1 only) increases rapidly after day 4 of adult emergence, while no CP is detected in male adults. In females CPs were detected only in the fat body, hemolymph and ovary. In the mid-5th-instar nymphs, CPs (CP-A and B) are mainly distributed in the hemolymph. CPs in the Hemolymph decrease during nymphal-adult ecdysis, whereas they increase in the fat body. CPs disappear from both the hemolymph and fat body by 2 days after ecdysis. Subsequently in the adult stage only CP-A increases again in the fat body and ovary. By tracer experiments using [³⁵S]-methionine, the fat body was shown to be the site of CP synthesis. CP-A and B synthetic activity was detected in nymphal females whereas, only CP-A synthesis was observed in adult females, while no CP synthesis was seen in adult males.     

Abnormally High Nourishment During Sensitive Periods Results in Body Weight Changes Across Generations

Objective : This study asked whether a brief period of over-nutrition during a developmentally sensitive time could impact the individual's adult weight and that of succeeding generations. Research Methods and Procedures : Female rat pups (F₁ generation) were randomly assigned to 1 of 3 groups: (1) a control group that was naturally reared by mothers; (2) another control group implanted with chronic gastric fistulas on postnatal day 4 and fed enough formula to match the growth of the mother-reared group; and (3) an experimental group gastrostomized and infused from day 8 through day 16 with a greater quantity of food than gastrostomy-reared controls (OF). On postnatal day 16, both gastrostomy-reared groups were returned to normal litters. Adult F₁ females from overfed and mother-reared groups were bred with normal males to yield an F₂ generation. F₂ adult females were bred to normal males to produce an F₃ generation. Results : When adult, the F₁ experimental group was heavier than control groups. F₂ adults from OF mothers were smaller than those from the control group. F₃ animals from OF grandmothers were heavier at weaning than F3 descendants from mother-reared animals. Discussion : Excess nourishment during a developmentally sensitive period changed the metabolic phenotype of one generation so dramatically that the gestational development and subsequent phenotype of two succeeding generations were also changed. The experiment models fetal effects of gestational diabetes in humans and may help to elucidate how, independent of genetic anomalies, secular changes can be detected across generations.     

Protective effect of esculin against prooxidant aflatoxin B1-induced nephrotoxicity in mice

The study was designed to investigate the protective effect of esculin against pro-oxidant aflatoxin B₁ (AFB₁)-induced nephrotoxicity in mice. In this study toxicity was developed by oral administration of AFB₁ at a dose of 66.60 μg/kg bw/day for 90 days in male Swiss albino mice. Esculin (150 mg/kg bw/0.2 ml/day) and standard compound ascorbic acid (300 mg/kg bw/0.2 ml/day) was given after 30 min of AFB₁ administration for 90 days. Protective efficacy was assessed by measuring the levels of lipid peroxidation (LPO) and non-enzymatic antioxidants such as reduced glutathione (GSH) and also by measuring activities of enzymatic antioxidants such as glutathione peroxidase (GPX), glutathione-S-transferase (GST), glutathione reductase (GR), superoxide dismutase (SOD) and catalase (CAT) in kidney. Results were analysed at the 30^th, 60^th and 90^th day of the daily treatments, which showed a decrease in the level of LPO and an increase in the levels of enzymatic and non-enzymatic antioxidants. The protective effect of esculin was further proved by histopathological findings as it exhibited regenerative activities in mice renal tubules against AFB₁-induced nephrotoxicity. The results obtained clearly demonstrate that the protective efficacy of esculin against pro-oxidant AFB₁-induced nephrotoxicity in mice might be due to its antioxidants and free radical scavenging properties.     

An in vitro study of alkaline phosphatase sensitivity to mixture of aflatoxin B<Subscript>1</Subscript> and fumonisin B<Subscript>1</Subscript> in the hepatopancreas of coastal lagoon wild and farmed shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

This study aimed to establish the combined effect of aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁) on wild Litopenaeus vannamei hepatopancreas alkaline phosphatase (AP) activity compared with that of farmed shrimp. AP activity in hepatopancreas extract was confirmed by several specific inhibitor assays. AP activity of wild shrimp was higher than that of farmed shrimp (p?<?0.05). However, AP activity from both wild and farmed shrimp was inhibited when incubated with AFB₁ and FB₁. The greatest inhibition occurred when AP was incubated with a mixture of AFB₁ and FB₁. The IC₅₀ for AFB₁ on AP activity of wild and farmed shrimp hepatopancreases was 0.790 and 0.398 μg/mL, respectively. The IC₅₀ of FB₁ was 0.87 μg/mL for wild shrimp and 0.69 μg/mL for farmed shrimp. These results suggest that, at the mycotoxins concentrations used in the study, AP from farmed L. vannamei was sensitive to the presence of both mycotoxins; however, AP is more sensitive to the combination of AFB₁?+?FB₁ suggesting a possible synergistic or potentiating inhibitory effect.     

A signal-amplified electrochemical immunosensor for aflatoxin B1 determination in rice

A sensitive and simple electrochemical immunosensor based on enzymatic silver deposition amplification was constructed for the detection of aflatoxin B₁ (AFB₁) in rice. The immunosensor was based on an indirect competitive format between free AFB₁ and aflatoxin B₁-bovine serum albumin (AFB₁-BSA) conjugate immobilized on the electrode surface for binding to a fixed amount of anti-AFB₁ antibody. Then the alkaline phosphatase (ALP)-labeled anti-mouse immunoglobulin G (IgG) secondary antibody was bound to the electrode surface through reaction with primary antibody. Finally, ALP catalyzed the substrate, ascorbic acid 2-phosphate, into ascorbic acid that reduced silver ions in solution to metal silver deposited onto the electrode surface. Linear sweep voltammetry was carried out to quantify the metal silver, which indirectly reflected the amount of the analyte. The experimental parameters, such as the dilution ratio of antibody and the concentration of AFB₁-BSA conjugate, have been evaluated and optimized. At the optimal conditions, the working range of the electrochemical immunosensor was from 0.1 to 10 ng/ml with a detection limit of 0.06 ng/ml. Good recoveries were obtained for the detection of spiked rice samples. So, the proposed method in this article could find a good use for screening AFB₁ in real samples.     

Ovarian sequestration of [14C]-inulin by an insect: An index of vitellogenesis

[¹⁴C]-Inulin injected into the blood of female milkweed bugs (Oncopeltus fasciatus) undergoing vitellogenesis is sequestered in the eggs. Determination of ovarian radiocarbon uptake gives a reproducible index of the progression of vitellogenesis that permits quantitative measurements prior to oviposition or under conditions where vitellogenesis is incomplete. This assay allows intra- and interspecific comparisons of the rates of juvenile hormone biosynthesis by corpora allata (CA) when these endocrine glands are transplanted into milkweed bug females, whose CA have been chemically destroyed with precocene. The method has the potential of distinguishing between nervous and hormonal regulation of the CA.     

Aflatoxin B1 affects feed consumption in male rats through an action on the central nervous system

Aflatoxins (AFTs), secondary metabolites of the biodeteriogens Aspergillus flavus and A. parasiticus, include aflatoxin B₁ (AFB₁), which is hepatocarcinogenic, can cause cellular and tissue damage and because its chemical composition is similar to that of certain steroids, may exhibit some pathological activities similar to those of steroids. The latter can suppress feed consumption by acting upon the central nervous system. Here, is reported the results of an investigation designed to assess whether this biodeteriogen could alter either feed consumption or plasma glucose levels. Male Sprague-Dawley rats maintained for two weeks upon laboratory rat chow were subjected to either intracerebroventricular (ICV) or intravenous (IV) injections of both AFB₁ and estradiol. Both non-injected and carrier-injected (0·9% NaCl) animals served as controls. Following fasting for 21 h, the rats were provided with a weighed amount of feed and both feed consumption and plasma glucose levels quantified during the 22nd, 23rd and 24th hour. Both ICV injections of 10 and 100 ng AFB₁ and IV injections of 1 and 10 μg AFB₁ kg⁻¹ significantly (p < 0·01) suppressed daily feed intake, but did not affect plasma glucose levels. This supports the hypothesis that AFB₁ suppresses feed intake probably through action upon the central nervous system. If true, this action as well as the toxic and carcinogenic aspects, further support the need for the prevention and/or removal of the mycotoxin from food and feed.     

Cyanoprotein: Developmental stage,sex and diapause-dependent expression,and synthesis regulation by juvenile hormone in the bean bug,Riptortus clavatus

Cyanoprotein (CP) synthesis was studied in nymphal and nondiapause adult, diapause adult, and juvenile hormone (JH) treated adult bean bugs, Riptortus clavatus. Hemolymph collected from bugs injected with [³⁵S]-methionine was analyzed by native polyacrylamide gel electrophoresis (PAGE) and fluorography, and CP synthesis was also determined quantitatively by rocket immunoelectrophoresis of hemolymph and counting. In the nymphal stages synthesis of CP-1, 2, 3, and 4 (with CP-4 synthesis predominant) reached a maximum at mid-instar and was not detected at each ecdysis, so that the synthetic activity of CPs changed cyclically in each instar. During the nymphal stages CP synthesis showed the same pattern and level in both females and males, and both diapause and nondiapause oriented bugs. In the adult stage, however, CP synthesis differed in the two sexes and nondiapause or diapause conditions. In nondiapause males CP synthesis was not detected in the adult, but in nondiapause females CP (only CP-1) was synthesized through the reproductive stages. CP-1 accumulated in the egg yolk together with vitellogenin. In diapause adults (both females and males) CP-1 to 4 were synthesized at a very low rate for over 2 months and accumulated in the hemolymph. JH or JH analog (JHA) methoprene and also long day condition, which terminate diapause, switched the main CP synthesis CP-4 to CP-1 in females. CP synthesis in diapause males stopped after JH(A) treatment. The activities of CP synthesis, thus, changed in developmental stages, sexes, and diapause. This is an excellent system for study of specific gene expression and switching controlled by insect hormones and sex. © 1992 Wiley-Liss, Inc.     

Seasonal variation in antifouling activity of crude extracts of the brown alga Bifurcaria bifurcata (Cystoseiraceae) against cyprids of Balanus amphitrite and the marine bacteria Cobetia marina and Pseudoalteromonas haloplanktis

Previous studies have demonstrated that macroalgae from Brittany (France) contain products with antifouling activity against marine bacteria, fungi, diatoms, seaweeds and mussels. Little is known regarding the ecological function of these compounds and insufficient attention has been paid to evaluating the possible temporal variation in antifouling activity. Studies of chemical defenses in both terrestrial and marine organisms suggest that organisms vary widely in the production of chemical defenses associated with physical (temperature, light) and biological (e.g. grazing pressure) factors, season and geographical location. The present study aimed to investigate the antifouling activity of crude extracts of monthly collections of the brown alga, Bifurcaria bifurcata, against two marine bacteria, Cobetia marina and Pseudoalteromonas haloplanktis, and cypris larvae of the barnacle, Balanus amphitrite. The toxicity of the extracts was determined with a B. amphitrite nauplius assay.The antimicrobial activity of the extracts was found to be subject to seasonal variation, with the highest level of activity recorded from samples collected between April and September. Results of the anti-settlement experiments showed that the extracts of B. bifurcata (when tested from 0 to 100 μg/ml) can be divided into three groups on the basis of their minimum inhibitory concentrations (MICs): (1) extracts from plants collected from September to March reduced settlement at nontoxic concentrations (50-100 μg/ml); (2) extracts from plants collected from April to July (which were the most active extracts) reduced settlement significantly when tested at >5 μg/ml, but were toxic at 100 μg/ml; (3) the extract prepared from plants harvested in August was inhibitory at >25 μg/ml, but was toxic at 100 μg/ml. Toxicity tests on nauplii showed that LC₅₀ values of samples from the September to March collections were >100 μg/ml, demonstrating that they were nontoxic to nauplii. In contrast, samples obtained from the April to August collections were toxic to nauplii; the most toxic ones being from algae collected in May (LC₅₀=55.6 μg/ml) and in June (LC₅₀=38.3 μg/ml).The antifouling activity of extracts thus reached a peak in summer corresponding to maximal values for water temperature, light intensity and fouling pressure. It remains to be investigated whether this activity has an ecological role in the alga.