Environmental Risk Assessment of Trifluoroacetic Acid

The Montreal Protocol was developed in 1987 in response to concerns that the chlorofluorocarbons (CFCs) were releasing chlorine into the stratosphere and that this chlorine was causing a depletion of stratospheric ozone over Antarctica. This international agreement called for a phase out of these CFCs. Industry initiated a major effort to find replacements that are safe when properly used and safe to the environment. The toxicology and environmental fate of these first generation replacements has been studied extensively. It was determined that the new substances break down in the environment to give predominantly carbon dioxide, water and inorganic salts of chlorine and fluorine. The only exception is that some substances also break down to yield trifluoroacetic acid (HTFA), a substance resistant to further degradation. Recognizing this, industry embarked on a research and assessment program to study the potential effects of trifluoroacetate (TFA) on the environment and to investigate possible degradation pathways. The results of these recently completed studies are summarized below and described in further detail in this paper. Trifluoroacetic acid is a strong organic acid with a pKa of 0.23. It is miscible with water and its low octanol/water partition coefficient (log P_ow=?2.1) indicates no potential to bioaccumulate. Industrial use is limited and environmental releases are very low. Some additional TFA will be formed from the breakdown of a few halogenated hydrocarbons, most notably HFC-134a (CF₃CH₂F), HCFC-124 (CF₃CHFCl), and HCFC-123 (CF₃CHCl₂). As these substances have only been produced in limited commercial quantities, their contribution to environmental levels has been minimal. Surprisingly, environmental measurements in many of diverse locations show existing levels of 100 to 300?ng·l^?1 in water with one site (Dead Sea) having a level of 6400?ng·l^?1. These levels cannot be accounted for based on current atmospheric sources and imply a long-term, possibly pre-industrial source. Generally, soil retention of TFA is poor although soils with high levels of organic matter have been shown to have a greater affinity for TFA when contrasted to soils with low levels of organic matter. This appears to be an adsorption phenomenon, not irreversible binding. Therefore, TFA will not be retained in soil, but will ultimately enter the aqueous compartment. Modeling of emission rates and subsequent conversion rates for precursors has led to estimates of maximum levels of TFA in rain water in the region of 0.1?µ·^?1 in the year 2020. TFA is resistant to both oxidative and reductive degradation. While there had been speculation regarding the possibility of TFA being degraded into monofluoroacetic acid (MFA), the rate of breakdown of MFA is so much higher than for TFA that any MFA formed would rapidly degrade. Therefore, there would be no buildup of MFA regardless of the levels of TFA present in the environment. Although highly resistant to microbial degradation, there have been two reports of TFA degradation under anaerobic conditions. In the first study, natural sediments reduced TFA. However, even though this work was done in replicate, the investigators and others were unable to reproduce it in subsequent studies. In the second study, radiolabeled TFA was removed from a mixed anaerobic in vitro microcosm. Limited evidence of decarboxylation has also been reported for two strains of bacteria grown under highly specific conditions. TFA was not biodegraded in a semi-continuous activated sludge test even with prolonged incubation (up to 84 days). TFA does not accumulate significantly in lower aquatic life forms such as bacteria, small invertebrates, oligochaete worms and some aquatic plants including Lemna gibba (duckweed). Some bioaccumulation was observed in terrestrial higher plants, such as sunflower and wheat. This result appeared to be related to uptake with water and then concentration due to transpiration water loss. When transferred to clean hydroponic media, some elimination of TFA was seen. Also, more than 80% of the TFA in leaves was found to be water ex-tractable, suggesting that no significant metabolism of TFA had occurred. At an exposure level of 1200?mg·l^?1 of sodium trifluoroacetate (NaTFA) — corresponding to 1000?mg·l^?1 HTFA — no effects were seen on either Brachy-danio rerio (a fish) or Daphnia magna (a water flea). With duckweed, mild effects were seen on frond increase and weight increase at the same exposure level. At a concentration of 300?mg·l^?1 no effects were observed. Toxicity tests were conducted with 11 species of algae. For ten of these species the EC50 was greater than 100?mg·l^?1. In Selenastrum capricornutum the no-effect level was 0.12?mg·l^?1. At higher levels the effect was reversible. The reason for the unique sensitivity of this strain is unknown, but a recovery of the growth rate was seen when citric acid was added. This could imply a competitive inhibition of the citric acid cycle. The effect of TFA on seed germination and plant growth has been evaluated with a wide variety of plants. Application of NaTFA at 1000?mg·l^?1 to seeds of sunflower, cabbage, lettuce, tomato, mung bean, soy bean, wheat, corn, oats and rice did not affect germination. Foliar application of a solution of 100?mg·l^?1 of NaTFA to field grown plants did not affect growth of sunflower, soya, wheat, maize, oilseed rape, rice and plantain. When plantain, wheat (varieties Katepwa and Hanno) and soya were grown in hydroponic systems containing NaTFA, no effects were seen on plantain at 32?mg·l^?1, on wheat (Katepwa) and soya at 1?mg·l^?1, or on wheat (Hanno) at 10?mg·l^?1; some effects on growth were seen at, respectively, 100?mg·l^?1, 5?mg·l^?1, 5?mg·l^?1, and 10?mg·l^?1 and above. TFA is not metabolized in mammalian systems to any great extent. It is the major final metabolite of halothane, HCFC-123 and HCFC-124. The half-life of TFA in humans is 16 hours. As expected, the acute oral toxicity of the free acid is higher than the one of the sodium salt. The inhalation LC₅₀ (2 hour exposure) for mice was 13.5?mg·l^?1 (2900?ppm) and for rats it was 10?mg·l^?1 (2140?ppm). Thus, TFA is considered to have low inhalation toxicity. The irritation threshold for humans was 54?ppm. As one would expect of a strong acid, it is a severe irritant to the skin and eye. When conjugated with protein, it has been shown to elicit an immunolog-ical reaction; however, it is unlikely that TFA itself would elicit a sensitization response. Repeat administration of aqueous solutions have shown that TFA can cause increased liver weight and induction of peroxisomes. Relative to the doses (0.5% in diet or 150?mg·kg^?1·day^?1 gavage) the effects are mild. In a series of Ames assays, TFA was reported to be non-mutagenic. Its carcinogenic potential has not been evaluated. Although TFA was shown to accumulate in amniotic fluid following exposure of pregnant animals to high levels of halothane (1200?ppm), no fetal effects were seen. Likewise, a reproduction study that involved exposure of animals to halothane at levels up to 4000?ppm for 4 hours per day, 7 days per week, resulted in no adverse effects. Given the high levels of halothane exposure, it is unlikely that environmental TFA is a reproductive or developmental hazard. Overall the toxicity of TFA has been evaluated in stream mesocosms, algae, higher plants, fish, animals and humans. It has been found to be of very low toxicity in all of these systems. The lowest threshold for any effects was the reversible effect on growth of one strain of algae, Selenastrum capricornutum, which was seen at 0.12?mg·l^?1. There is a 1000-fold difference between the no-effect concentration and the projected environmental levels of TFA from HFCs and HCFCs (0.0001?mg·l^?1). Based on available data, one can conclude that environmental levels of TFA resulting from the breakdown of alternative fluorocarbons do not pose a threat to the environment.     

Effects of pH on toxicity of As,Cr, Cu,Ni and Zn to Selenastrum capricornutum Printz

Acute toxicity tests were conducted to establish the response of Selenastrum capricornutum Printz to sublethal concentrations of As, Cr, Cu, Ni and Zn at a broad range of pH levels. Cultures were incubated for a period of seven days at pH 4 in standard algal assay media containing sublethal concentrations of metals. At this low pH, growth was depressed for all metals tested. The adjustment of pH to higher levels resulted in increased growth when cultures were treated with As, Cu, or Ni and incubated for an additional 7 days. Toxicity was least at the optimum pH range for growth of the alga.The observation that the toxicity of As, Cu, and Ni to S. capricornutum decreases markedly at pH values above 4.0 may be of ecological importance in the control of acid mine pollution. If a high percentage of algae show a similar response to decreasing toxicity with increasing pH, it clearly would be of value to adopt measures which control pH as well as the levels of metals present. It was suggested that algae with a broad pH growth range, such as S. capricornutum, could benefit from the addition of highly alkaline materials to waters where certain metals are present.     

Effects of the novel allelochemical ethyl 2-methylacetoacetate from the reed (<Emphasis Type="Italic">Phragmitis australis</Emphasis> Trin) on the growth of several common species of green algae

Bioassays were performed to investigate the effects of the novel allelochemical, ethyl 2-methylacetoacetate (EMA), isolated from the reed (Phragmitis australis) on the growth of three common species of algae; Scenedesmus obliquus, Selenastrum capricornutum and Chlamydomonas reinhardtii. The results demonstrated that EMA has three quite different types of effect on these three species of algae. The growth of S. capricornutum was significantly inhibited by EMA during the whole cultivation period. The EC₅₀ values of EMA on S. capricornutum was 0.6 mg L⁻¹(7 days). However, the inhibitory effect of EMA on S. obliquus was apparent during the first 4 days of batch cultivation and then the inhibitory effect disappeared, and a stimulating effect was observed instead. The EC₅₀ value of EMA on S. obliquus was 0.43 mg L⁻¹(4 days). In addition, following the addition of EMA, the cells of S. obliquus and S. capricornutum became significantly larger than the normal untreated one and the algal cells changed morphologically. The microstructure of the algal cells was disrupted by the addition of EMA. There was no significant inhibition of the growth of C. reinhardtii by EMA, but cell motility was affected.     

Intermittent lead‐induced stress on antioxidant enzyme activity and subcellular distribution of Pb in Pogonatherum crinitum seedlings

• Pogonatherum crinitum is a promising lead (Pb) hyperaccumulator due to its high Pb tolerance and accumulation ability. However, the mechanisms that support Pb accumulation and tolerance in P. crinitum are not yet clearly understood.
• An indoor hydroponic experiment was conducted by cultivating P. crinitum seedlings exposed to intermittent Pb stress for 60 days, divided into four stages (T1, T2, T3 and T4), with a 15‐day duration per stage. The following concentrations of Pb were used: 0, 500, 0, 500 mg·l^?1 and 0, 1000, 0, 1000 mg·l^?1). Antioxidant enzyme activity, Pb concentration and subcellular distribution of Pb were measured at each of the above stages.
• The results showed that superoxide dismutase (SOD) activity in shoots, and SOD, peroxidase (POD) and malondialdehyde (MDA) activity in shoots and roots significantly increased from T1 (no Pb stress) to T2 (Pb stress) in both 500 mg·l^?1 and 1000 mg·l^?1 treatments; however, no significant difference was noted between stages T3 (no Pb stress) and T4 (Pb stress). There was no obvious effect of Pb stress on catalase (CAT) activity in shoots and roots among different stages. The Pb concentration in shoots was up to 5090.90 mg·kg^?1 and 7573.57 mg·kg^?1, and the bioconcentration factor (BFC) was 10.18 and 7.57 for the 500 mg·l^?1 and 1000 mg·l^?1 treatments, respectively, which confirmed the Pb hyperaccumulator characteristics of P. crinitum. For plants under Pb stress, most of the Pb was fixed in the cell walls, with a smaller amount in leaves and root vacuoles.
• Both SOD and POD scavenging of reactive oxygen radicals and fixing and compartmentalisation of Pb in the cell wall might play important roles in detoxification of P. crinitum seedlings in response to Pb stress. There was no phased response of P. crinitum to intermittent Pb stress and the physiological response to Pb stress may be contiguous.

     

PHOSPHATE-LIMITED GROWTH KINETICS OF SELENASTRUM CAPRICORNUTUM (CHLOROPHYCEAE)1

Most theoretical studies of phytoplankton growth in aquatic environments assume that relative nutrient utilization abilities regulate species composition. The steady-state phosphate-limited growth kinetics of Selenastrum capricornutum Printz were examined using continuous cultures to characterize the green alga's ability to compete for orthophosphate (Pi) when Pi limits growth. The maximal specific growth rate for Selenastrum at 20 C was 1.20 day^?1, and the concentration where half maximal growth rate occurs was 40 nM Pi. There was an apparent threshold of 10 nM Pi. Cell yields varied inversely with growth rate; thus ability to utilize Pi could not be characterized in terms of the Monod half-saturation constant and maximal growth rate. Instead, we computed the Pi affinity from steady-state flux vs. external Pi concentrations. This affinity was 2.8 l·mg dry wt^?1· day^?1 for Selenastrum. Kinetic evidence from this study suggests that Selenastrum will not be growth competitive with some other common aquatic heterotrophs and autotrophs when Pi limits microbial growth in lakes.     

Molybdate reduction by Pseudomonas sp. strain DRY2

Aims: To isolate and characterize a potent molybdenum‐reducing bacterium. Methods and Results: A minimal salt medium supplemented with 10 mmol l^?1 molybdate, glucose (1·0%, w/v) as a carbon source and ammonium sulfate (0·3%, w/v) as a nitrogen source was used in the screening process. A molybdenum‐reducing bacterium was isolated and tentatively identified as Pseudomonas sp. strain DRY2 based on carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny. Strain DRY2 produced 2·4, 3·2 and 6·2 times more molybdenum blue compared to Serratia marcescens strain DRY6, Enterobacter cloacae strain 48 and Eschericia coli K12, respectively. Molybdate reduction was optimum at 5 mmol l^?1 phosphate. The optimum molybdate concentration that supported molybdate reduction at 5 mmol l^?1 phosphate was between 15 and 25 mmol l^?1. Molybdate reduction was optimum at 40°C and at pH 6·0. Phosphate concentrations higher than 5 mmol l^?1 strongly inhibited molybdate reduction. Inhibitors of electron transport system such as antimycin A, rotenone, sodium azide and cyanide did not inhibit the molybdenum‐reducing enzyme activity. Chromium, copper, mercury and lead inhibited the molybdenum‐reducing activity. Conclusions: A novel molybdenum‐reducing bacterium with high molybdenum reduction capacity has been isolated. Significance and Impact of the Study: Molybdenum is an emerging global pollutant that is very toxic to ruminants. The characteristics of this bacterium suggest that it would be useful in the bioremediation of molybdenum pollutant.     

ETHEPHON (2-CHLOROETHYLPHOSPHONIC ACID) EFFECTS ON SCENEDESMUS QUADRICAUDA (CHLOROPHYCEAE)1,2

Growth of Scenedesmus quadricauda (Turp.) Bréb. was significantly increased when the cultures were grown in Ethephon 68–241 (2-chloroethylphosphonic acid) at concentrations of 0.001–1.0 μg · ml^?1. At 0.1 μg · ml^?1, Ethephon caused a significant increase in RNA levels on a dry weight basis. Total protein and DNA levels increased but not significantly. These data indicate that ethylene, the decomposition product of Ethephon at physiological pH, has some effect on the metabolism of algae.     

Grazing resistance in nutrient-stressed phytoplankton

Grazing experiments were performed with the zooplankters Daphnia pulex and Daphnia magna, feeding on phosphorus-saturated and phosphorus-limited cells of two green algae (Scenedesmus subspicatus and Selenastrum capricornutum). P-limited algal cells passed largely intact through the gut and were thus spared from heavy grazing pressure. P-saturated algal cells, in contrast, were efficiently assimilated. Structural and morphological changes in the P-limited cells most probably reduced their digestibility. This phenomenon may be an important factor in zooplankton production and competition, and may serve as an example of a highly efficient strategy of P-limited algae to resist heavy grazing pressure.     

METHYLAMINE UPTAKE IN THE GREEN ALGA CHLORELLA PYRENOIDOSA1

Methylamine uptake in nitrogen-starved Chlorella pyrenoidosa Beij. follows Michaelis-Menten kinetics: maximum uptake is about 1.6 nmol μl^?1· cells · min^?1, half-saturation occurs at 4 μM methylamine, and the slope in the range where uptake is proportional to concentration is 0.4 nmol μl^?1· min^?1·μM^?1. In cells grown in the presence of a non-limiting nitrogen concentration, methylamine uptake is directly proportional to concentration up to at least 0.5 mM, and the slope is 1/500 that for starved cells. Similar uptake kinetics have been reported for Penicillium chrysogenum and attributed to an inducible “ammonium permease.” Apparently, a similar permease occurs in algae.     

MODULATION OF PHOTOSYNTHETIC CARBON ASSIMILATION IN SELENASTRUM CAPRICORNUTUM (CHLOROPHYCEAE) BY cAMP: AN ELECTROGENIC MECHANISM?1

Laboratory batch cultures of the unicellular alga Selenastrum capricornutum Printz (Chlorophyceae) were used as a model system to test the hypothesis that the regulatory molecule cAMP may alter algal photo synthetic carbon assimilation (PCA) rates via an electrogenic mechanism. In one series of experiments, cells were resuspended in cAMP-free media subsequently augmented with 0.5 nmol·L^?1to 0.1 μmol·L^?1of added cAMP, concentrations paralleling dissolved cAMP levels in lake water and culture filtrates but insufficient to result in short-term increases in intracellular cAMP content. cAMP-treated cells were moderately and transiently hypopolarized concomitant with a reduction in PCA capacity. Media and intracellular pH and quinacrine fluorescence quenching rates were also altered, with similar effects on PCA, but specific effects appeared to be associated with diel cycles. Higher concentrations of added cAMP (10 μmol·L^?1to 10 mmol·L^?1) further hypopolarized the transmembrane potential, greatly enhanced PCA rates, acidified the cell interior, and enhanced quinacrine quenching rates. With the exception of cGMP, no nucleotide derivative examined elicited the same effects. Inhibitors of plasmalemma ATPase and NADH oxidase activity also hypopolarized the membrane potential but inhibited PCA rates. Collectively, the data support the cAMP-electrogenic thesis, but the mechanism, although resembling features of H⁺/ ion-cotrans-port, may in fact be more complex.     

Synergistic effect of DFMO and 2,4-D on regeneration of Tabernaemontana persicariaefolia jacq in vitro

Callus cultures of Tabernaemontana persicariaefolia, (Apocynaceae), an endangered species endemic to the Mascarene Islands, were established from leaf explants on MS medium containing either 5 mg·l⁻¹ 2,4-D and 0.5 mg·l⁻¹ BA or 5 mg·l⁻¹ 2,4-D, 0.5 mg·l⁻¹ BA and 200 mg·l⁻¹ DFMO. Histological studies showed regenerating nodules resembling globular embryos in calli after 4 weeks on the DFMO medium. Green shoot formation was achieved by sequential subculture of the induced calli on media with gradually decreasing 2,4-D concentrations (5→1→0 mg·l⁻¹). Regeneration was greatly stimulated in the presence of DFMO. The first emergence of shoots occured 3 weeks earlier than in untreated callus cultures.     

Micropropagation studies inCeropegia SPP

Summary The purpose of this study was to developin vitro techniques for conserving wild and endemic species ofCeropegia by mass multiplication for subsequent reintroduction in their natural habitat. Micropropagation involving a combination of axillary bud culture, shoot multiplication, somatic embryogenesis andin vitro tuber formation forCeropegia jainii, a rare plant of the Indian sub continent,C. bulbosa var.bulbosa andC. bulbosa var.lushii, common species, was developed. Nodal explants from all species were cultured on 0.5 MS medium with 8.8 μM (2 mg·l⁻¹) N⁶-benzyl aminopurine (BA) to regenerate the axillary buds. These produced multiple shoots when transferred to multiplication medium consisting of 0.5 MS medium with 2.2 μM (0.5 mg·l⁻¹) BA, or microtubers when transferred to 0.5 MS medium with 22.2 μM (5 mg·l⁻¹) BA and 23.2 μM (5 mg·l⁻¹) kinetin.In vitro flowering occurred inC. jainii and not in the other two varieties when the plants were cultured on multiplication media with spermine at 0.25 μM (50 μg·l⁻¹) as an additive. Shoot pieces produced callus on MS medium with 9.05 μM (2 mg·l⁻¹) 2,4-dichlorophenoxy acetic acid. Regeneration of the calli by somatic embryogenesis was achieved when they were transferred to 0.5 MS medium with 2.2 μM (0.5 mg·l⁻¹) BA. Rooting of the shoots was possible both byin vitro andex vitro means.     

Metal-ion susceptibility of oral bacterial species

Aims: This study aimed to evaluate the effect of lead (Pb) on growth of bacterial species related to dental diseases in vitro. Methods and Results: The effects of lead acetate on representative species of the oral flora were examined at 0·1–10 mmol l^?1 and compared with the effect of silver nitrate and ferrous sulfate. The minimal inhibitory concentration of lead acetate was between 0·15 and 5 mmol l^?1 for the bacterial strains tested. The minimal bactericidal concentration of lead acetate for most oral species was detected in the range of 5–10 mmol l^?1. Silver nitrate at a concentration of 1·25 mmol l^?1 was sufficient to exhibit antibacterial activity against almost all bacteria tested. Ferrous sulfate had the lowest effect. Conclusions: The study indicated a general antimicrobial effect of lead on oral bacterial species in the range of 0·15–10 mmol l^?1. The toxicity of silver nitrate was the highest, whereas that of ferrous sulfate was the lowest. Gram‐positive species had a tendency to be less susceptible for metals than Gram‐negatives. Significance and Impact of the Study: The study shows that it is possible that microbiological changes may occur in the dental plaque in children because of toxic exposure of environmental lead.     

Hygromycin promotes somatic embryogenesis in spinach

Hygromycin (hyg) at low doses (0.5–1.0 mg l^?1) promoted somatic embryogenesis from apical sections of spinach lateral roots. The highest promoting effect on both the frequency of regeneration and the mean number of somatic embryos (SE) per explant was achieved at 0.5 mg l^?1 hyg. With increasing the concentration of hyg to 1 mg l^?1, the regeneration frequency decreased, while the mean SE number remained significantly higher than in control (hyg-free medium). Complete inhibition of SE regeneration started at 7.5 mg l^?1 hyg. Moreover, hyg efficiently promoted the process of secondary somatic embryogenesis. Compared to control, a 2.75-fold increase in the secondary somatic embryo (SSE) mean number was obtained at 0.5 mg l^?1 hyg, and the increment was still discernible at 1.0 and 2.5 mg l^?1 hyg. Both primary SE and SSE explants became completely necrotic at 12.5 mg l^?1 hyg. Since attempts with direct selection at 20 mg l^?1 hyg proved unsuccessful, the results obtained in this study suggest that a stepwise selection procedure is suitable, starting with selection at 0.5 mg l^?1 hyg, to exploit the promoting effect of low hyg doses on SE regeneration from transformed cells, then gradually increasing the hyg concentration to 20 mg l^?1 for final selection. Complete SE and SSE explant mortality at hyg above 12.5 mg l^?1 guarantees a low possibility of escape during the selection process. This study will be useful for increasing the efficiency of transgenic plant regeneration following genetic transformation in spinach.     

Enantioselective toxic effects of hexaconazole enantiomers against Scenedesmus obliquus

Enantioselectivity in ecotoxicity of chiral pesticides in the aquatic environment has been a subject of growing interest. In this study, the toxicological impacts of hexaconazole enantiomers were investigated with freshwater algae Scenedesmus obliquus. After 96 h of exposure, the EC₅₀ values for rac‐hexaconazole, (+)‐hexaconazole, and (?)‐hexaconazole were 0.178, 0.355, and 0.065 mg l^?1, respectively. Therefore, the acute toxicities of hexaconazole enantiomers were enantioselective. In addition, the different toxic effects were evaluated when S. obliquus were exposed to 0.2, 0.5, and 1.0 mg l^?1 of rac‐hexaconazole, (+)‐hexaconazole, and (?)‐hexaconazole during 96 h, respectively. The chlorophyll a and chlorophyll b contents of S. obliquus treated by (?)‐hexaconazole were lower than those exposed to (+)‐hexaconazole, whereas the malondialdehyde contents of S. obliquus treated by (?)‐form were higher than those exposed to (+)‐form at higher concentrations. In general, catalase activities were significantly upregulated by exposure to (?)‐enantiomer than (+)‐enantiomer at all three concentrations. However, superoxide dismutase activities exposed to (?)‐hexaconazole were lower than that exposed to (+)‐hexaconazole at 0.2 mg l^?1 and 0.5 mg l^?1. On the basis of these data, the acute toxicity and toxic effects of hexaconazole against S. obliquus were enantioselective, and such enantiomeric differences must be taken into consideration in pesticide risk assessment. Chirality 24:610–614, 2012. © 2012 Wiley Periodicals, Inc.     

Nutritional suitability of some uni-algal diets for freshwater calanoids: unexpected inadequacies of commonly used edible greens and others

• 1 Naupliar and copepodid development times (Dn and Dc, respectively) of two African freshwater calanoids (Metadiaptomus meridianus and Tropodiaptomus spectabilis) were measured on mono-specific diets of comparably sized Chlamydomonas reinhardii, Scenedesmus acutus, Cryptomonas sp., Rhodomonas minuta, Cyclotella meneghiniana, and Selenastrum capricornutum, to test the nutritional adequacy of these algae. Comparisons were made at a standard temperature (17°C) and food supply level (1 mgCl^?1).
• 2 All diets other than Scenedesmus and Selenastrum supported complete naupliar development at broadly comparable times within and between calanoids, apart from greatly protracted Dn values for M. meridianus on Cyclotella. Dc durations were more variable between diet types, and both Chlamydomonas and Cyclotella were inferior or inadequate for copepodid development.
• 3 Both naupliar and copepodid stages ingested radiolabelled Scenedesmus and Selenastrum readily. Comparative incorporation rate measures of Selenastrum and Cryptomonas respectively exceeded estimated metabolic maintenance needs of stage 3/4 nauplii of T. spectabilis by some 56% and 790%. Scope for growth (‘surplus’ energy) was accordingly fourteen-fold greater on Cryptomonas than on Selenastrum /Scenedesmus. The dietary inadequacy of these two green algae is thus attributed largely to low digestibility, and perhaps some biochemical deficiency.

     

Toxicité aiguë d'un fongicide dithiocarbamate,le thirame,chez des larves de l'ephémère Cloeon dipterum: effets de divers paramètres

Acute toxicity and degradation of an aqueous suspension of Thiram were studied in Cloeon dipterum. Lethal concentrations for 50% of the larvae (LC 50) after 24 to 96 h of exposure and thresholds were calculated. LC 50 varied from 0.39 to 1.92 mg·l^?1 according to the time and the physiological state of the larvae. It was demonstrated that:

the sensitivity of the larvae increased with the time during which they were kept in the laboratory;

while the toxicity of the aqueous suspension (1 mg·l^?1) of Thiram decreased with time, another toxic product was formed.

     

The algae-lytic ability of bacterium DC10 and the influence of environmental factors on the ability

A lysing-bacterium DC₁₀, isolated from Dianchi Lake of Yunnan Province, was characterized to bePseudomonas sp. It was able to lyse some algae well, such asMicrocystis viridis, Selenastrum capricornutum, and so on. In this study, it was shown that the bacterium lysed the algae by releasing a substance; the best lytic effects were achieved at low temperatures and in the dark. Different concentrations of CaCl₂ and NaNO₃ influenced the lytic effects; the ability to lyse algae decreased in the following order: pH 4 > pH 9 > pH 7 > pH 5.5. It was significant to develop a special technology with this kind of bacterium for controlling the bloomforming planktonic microalgae.     

Antioxidant responses of different microalgal species to nonylphenol-induced oxidative stress

The antioxidant responses of four green microalgae, i.e., Chlorella vulgaris, Chlorella sp., Selenastrum capricornutum and Scenedesmus quadricauda, under control, low (0.1 mg L^?1) and high (1.0 mg L^?1) nonylphenol (NP) concentration were studied. The antioxidant responses of microalgae to NP depended on both NP concentrations and exposure time. The effects of NP on antioxidant responses were most obvious on the first day of exposure and the effects decreased with prolonged exposure time. At low NP concentration, there were no significant changes in activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), or in glutathione (GSH) content, in all four species, while high concentration of NP led to different changes in these parameters. In NP-tolerant species, i.e., C. vulgaris and Chlorella sp., activities of SOD, CAT and POD increased remarkably when exposed to high NP concentration, while the increase was less evident or insignificant in Se. capricornutum and Sc. quadricauda, the two NP-sensitive species. On the other hand, the malondialdehyde (MDA) content declined gradually with increase in NP concentrations, particularly in C. vulgaris and Chlorella sp. Similarly, NP exposure caused an inhibition of glutathione peroxidase (GPX) activity in all four species. However, the changes of glutathione reductase (GR) and glutathione S-transferase (GST) activity did not seem to correlate with the NP tolerance of microalgae. These results suggested that various antioxidant mechanisms were involved in microalgae when exposed to NP, and the NP-tolerant species displayed more evident and rapid changes in some antioxidant responses than the NP-sensitive ones.     

BIOSYNTHESIS OF POLY‐3‐HYDROXYBUTYRATE (PHB) IN THE TRANSGENIC GREEN ALGA CHLAMYDOMONAS REINHARDTII1

A cell‐wall deficient strain of Chlamydomonas reinhardtii P. A Dang. CC‐849 was cotransformed with two expression vectors, p105B124 and pH105C124, containing phbB and phbC genes, respectively, from Ralstonia eutropha. The transformants were selected on Tris‐acetate‐phosphate media containing 10 μg · mL^?1 Zeomycin. Upon further screening, the transgenic algae were subcloned and maintained in culture. PCR analysis demonstrated that both phbB and phbC genes were successfully integrated into the algal nuclear genome. Poly‐3‐hydroxybutyrate (PHB) synthase activity in these transgenic algae ranged from 5.4 nmol · min^?1 · mg protein^?1 to 126 nmol · min^?1 · mg protein^?1. The amount of PHB in double transgenic algae was determined by gas chromatography–mass spectrometry (GC–MS) when comparing with PHB standard. In addition, PHB granules were observed in the cytoplasm of transgenic algal cells using TEM, which indicated that PHB was synthesized in transgenic C. reinhardtii. Hence, results clearly showed that producing PHB in C. reinhardtii was feasible. Further studies would focus on enhancing PHB production in the transgenic algae and targeting the chloroplast for PHB accumulation.