Chlamydomonas (Green algae: formerly including protozoa)

Chlamydomonas moewusii
Chlamydomonas reinhardtii

1. Chlamydomonas moewusii

[ref. ID; 799]

Test system

Cellular response to thermal and photo stress

Strains

Axenically.

Stress

Temperature 20, 25, 30, 32.5, and 35 degrees C. Light intensity 15, 60, and 150 foot-candles.

Test design

Chlamydomonas was inoculated from a 12-day culture and grown in 50 ml of organic medium (TPP) per 125-ml Erlenmeyer flask. Each flask was stirred one or twice each day.

Measurements

Cell counts.

2. Chlamydomonas reinhardtii

[ref. ID; 1319]

Test system

Growth inhibition

Strains

11-3Qa (from Sammlung von Algenkulturen, University of Gettingen, D-3400 Gottingen).

Toxicants

Atrazine, 3,4-Dichloroaniline, 1,2-Dichloropropane, Methylparathion, Lindane, Pentachlorophenol, Cu2+ (CuSO4/5H2O), Cd2+ (CdCl2/2.5H2O), Mg2+ (MgCl2).

Test design

OECD Guideline 201 (1984) & Flowthrough System. SAG medium 12, shaking. Temperature 20+/-1 degrees C. Photoperiod: Continuous uniform illumination (8000 lux, cool white fluorescent tube).

Measurements/observations

Growth rate, effective photosynthesis rate (ERP).

Evaluations

NOEC, EC10, EC50.

[ref. ID; 3904]

Test system

Flagellar activity test

Strains

137c, mating type +.

Toxicity

NiCl2 & NiCl2 + CaCl2, EDTA & EDTA + CaCl2.

Temperature

25 degrees C.

Measurements/observations

Motility, swimming speed, geotaxis, phototaxis, pattern swimming.

[ref. ID; 4534]

Test system

Photosynthesis inhibition

Strains

Wild type & Metribuzin-resistant mutants (MZ-1, MZ-2, MZ-3, MZ-4).

Toxicants

Atrazine, DCMU, metribuzin.

[ref. ID; 4553]

Test system

Photosynthesis inhibition

Strains

Wild type & Metribuzin-resistant mutants of C. reinhardtii (MZ-1, MZ-2 and MZ-4).

Toxicants

Atrazine, metribuzine, metamitron, DCMU (diuron), metabenzthiazuron, benzthiazuron, cyanoacrylate, ioxynil, dinoseb, bromonitrothymol, ketonitrile, tetrabromopyridinol.

Evaluations

I50-values.

[ref. ID; 4556]

Test system

Photosynthesis inhibition

Strains

Wild type & Metribuzin-resistant mutants of C. reinhardtii (MZ-1, MZ-2 and MZ-4).

Toxicants

2-bromo-4-nitrophenols, 2,4-dinitrophenols, 6-alkyl-4-nitrophenols, 2-bromo-4-nitro-6-alkylphenols.

Evaluations

I50-values.

[ref. ID; 4557]

Test system

Photosynthesis inhibition

Strains

Wild type & mutations of resistance to a variety of PS II herbicides.

Toxicants

DCMU.

[ref. ID; 4558]

Test system

Photosynthesis inhibition

Strains

Wild type CC-407 & strain RS-3 resistant to N-phenylimide herbicides.

Toxicants

Acifluorfen-ethyl, DCMU, N-phenylimide herbicides (S-23031: pentyl 2-chloro-4-fluoro-5-[(3,4,5,6,-tetrahydro)phthalimido]-phenoxyacetate, S-23121: N-[4-chloro-2-fluoro-5-[(1-methyl-2-propynyl)oxy]phenyl]-3,4,5,6-tetrahydrophthalimide, S-23142: N-[4-chloro-2-fluoro-5-propargyloxyphenyl]-3,4,5,6-tetrahydrophthalimide, S-53482: 7-fluoro-6-[(3,4,5,6,-tetrahydro)phthalimido]-4-(2-propynyl)-1,4-benzoxazin-3(2H)-one).

Evaluations

I90.

[ref. ID; 4565]

Test system

Microtiter growth assay

Strains

From the Chlamydomonas Genetics Center, Box 91000, Duke University, Durham, NC, 27708-1000 [wild type strain CC-125, the transformed mutant strain CC-2827 dr-1 (dr = DCMU resistant, psbA Ser264 transfer Ala)].

Toxicants

Atrazine, bromacil, DCMU, Metribuzin.

Temperature

25 degrees C.

Measurements/observations

Total biomass (spectrophotometrically at 750 nm).

Evaluations

I50, MLHC.

[ref ID; 4955]

Test system

The extracellular sorption of PCBs and the intracellular uptake in thylakoids (i.e. photosynthetic membranes)

Strains

Clamydomonas reinhardtii UTEX 89 and UTEX 2337.

Toxicants

PCBs (2-Chlorobiphenyl, 2,4'-Dichlorobiphenyl, 2,2',5,5'-Tetrachlorobiphenyl, 2,2',6,6'-Tetrachlorobiphenyl, 2,3,4,5-Tetrachlorobiphenyl, 3,3',4,4'-Tetrachlorobiphenyl, 3,3',4,4',5-Pentachlorobiphenyl, 2,2',3,3',4,4'-Hexachlorobiphenyl, 2,2',4,4',5,5'-Hexachlorobiphenyl, 2,2',4,4',6,6'-Hexachlorobiphenyl, 3,3',4,4',5,5'-Hexachlorobiphenyl, 2,2',3,3',4,4',5,5'-Octachlorobiphenyl, Decachlorobiphenyl).

Test design

Batch culture, 14:10-hr light-dark cycle (20,000 lux; 390 umol/m2s-1), temperature 25 degrees C (the extracellular sorption studies) and 8-10 degrees C (the thylakoids uptake studies).

[ref. ID; 4995]

Test system

The influence of different nutrient regimes on the accumulation of Cd, Zn, and Se(IV) over a 4 hr exposure period

Strains

From the Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan. Axenic culture (WC medium).

Toxicants

Radiotracers [109]Cd, [65]Zn, [75]Se(IV).

Test design

Phosphate concentration (0.1, 1.0, 10.0 uM) and Nitrate concentration (5, 40, 200 uM). Short-term exposure 4 hr.

Measurements/observations

Intracellular metals in algae.

[ref. ID; 6091]

Test system

Toxicants/concentrations

Anionic, Non-ionic, and Amphoteric Surfactants (0.02-2.0 mM).

Test design

21+/-2 degrees C, 18-hr photoperiods, 6 replicates.

Measurements

Optical density (absorption at 652 nm).

[ref. ID; 6096]

Test system

The effect on growth rate (8 days), photosynthesis rate and chlorophyll-a synthesis

Strains

C. reinhardtii (mt +) provided by Dr K.Y. Chan, Department of Biology, The Chinese University of Hong Kong, was originally obtained from University of Texas at Austin Culture Collection.

Toxicants/concentrations

2,4-D (1, 5, 10, 20, 40 ppm), Diazinon (1, 5, 10, 20, 40 ppm), Dimethoate (1, 5, 10, 20, 40 ppm), Fenitrothion (1, 5, 10, 20, 40 ppm), Malathion (1, 5, 10, 20, 40 ppm), Phenthoate (1, 5, 10, 20, 40 ppm), Quinalphos (1, 5, 10, 20, 40 ppm).

Test design

250 ml cotton-plugged Erlenmeyer flasks (MCM medium 18 ml + algal inoculation 2ml) which were placed in a gyratory shaker operated at 100 rpm, 25+/-2 degrees C, cool white fluorescent tubes with a light intensity of 6000 lux and a 16/8 hr light and dark cycle, duplicates.

Measurements

Absorbance using by spectrophotometer at 650 nm, chlorophyll-a, gross photosynthetic rate using by a Gilson Differential Respirometer.

[ref. ID; 6723]

Test system

Gene expression (global transcriptome analysis: Differential display analysis and Micoarray analysis)

Strains

Wild type C137.

Toxicants

Free Cd (Cd2+).

Test design

Differential display analysis: Cell exposed to the Tris-acetate-phosphate (TAP) medium including non-mental (control), 0.03 uM Cd2+, 0.34 uM Cd2+, 3.40 uM Cd2+, 0.50 uM Ni2+, and 0.17 uM Cu2+ for 2 hr under light.
Microarray analysis: 0.34 uM Cd+ in TAP medium for 2 hr.
Real-time quantitative polymerase chain reaction analysis (to find Cd-responsive genes as biomarker for Cd risk): Cell exposed to the TPA medium including nonmetal (control), 7.8x10E-3 uM, 7.1x10E-3 uM, 1.2 uM, and 9.0 uM for 2 hr under light.
Bioaccumulation (to determine the amount of intracellular Cd): Cell exposed to the TPA medium including non-metal (control), 7.8x10E-3 uM, 7.1x10E-3 uM, 1.2 uM, and 9.0 uM for 2 hr under light.

[ref. ID; 6926]

Test system

Genotoxicity

Strains

Wild-type C. reinhardtii (derived from a stock cultured at AstraZeneca). Cell wall-free C. reinhardtii were provided courtesy of Saul Purton and were the CW15.J3 strain, mating type minus, derived from a stock cultured at the University College London, United Kingdom, originally established by Jacqueline Girard-Bascou in France.

Toxicants

4-nitroquinoline-1-oxide (1 and 5 nM), N-hydroxy metabolite of 2-acetylaminofluorene (N-OH-2-AAF) (0.05 and 5 uM), and chrysoidine (0.1 and 10 uM).

Test design

Compound exposure was undertaken in three replicate flasks per exposure as follows. 50 ml of algal cells in mid-log phase (day 5) were collected by low-speed centrifugation (300 g, 10 min, 4 degrees C), and the pellet was resuspended in 20 ml of culture medium (freshwater algal culture medium FM/4). 1.98 ml of cell suspension were incubated with 20 ul chemicals. Temperature at 20+/-1 degrees C under a 12:12-hr light:dark cycle. Exposure period 24 hr.

Measurements/observations

DNA strand breaks (Comet assay).
Ethoxyresorufin-O-deethylase (EROD) activity.

[ref. ID; 6930]

Test system

Glutathione response

Strains

No. 90, mating type plus were obtained from the UTEX Culture Collection of Algae at the University of Texas.

Toxicants

[65]Cu (0, 10, 20, 40, 60, 80, and 100 nM total Cu), [111]Cd (0, 10, 20, 40, 60, 80, and 100 nM total Cd).

Test design

All experiments were carried out in acid-leached 500 ml polycarbonate Erlenmeyer flasks (Corning) under a high-efficiency particulate air (HEPA) filter in polycarbonate hoods in a temperature- and light-controlled room at 21.0+/-1 degrees C under continuous light (70+/-10 uE/m2/sec). Humic acid was added to several experimental flasks at 0.5 mg/L and 1.0 mg/L concentrations. Exposure period 6 and 24 hr.

Measurements/observations

Cell number, chlorophyll a, glutathione (total glutathione (TGSH) and oxidized glutathione (GSSG)), glutathione reductase enzyme activity.

Evaluations

Specific growth rate.

[ref. ID; 6996]

Test system

Cadmium-tolerance population

Strains

Wild-type strains, wall-defiecient mutant CW-15 and cadmium-tolerant CW-15Cd.

Toxicants

[109]CdCl2

Test design

CTM medium (0.5 mM MgCl2, 0.5 mM CaCl2, 7.5 mM (NH4)H2PO4, 7.5 mM KCl, 5 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)] (pH 6.3), 2% sodium acetate, and trace elements according to Starr).

Measurements/observations

Cell dencity, chlorophyll concentrations, starch content, cadmium-binding peptides, protein concentrations.

[ref. ID; 7001]

Test system

The influence of the cell wall and pH

Strains

TWo axenic strains of Chlamydomonas reinhardtii (UTCC 11, walled wild type and UTCC 12, cell wall-less mutant) were obtained from the University of Toronto Culture Collection (UTCC).

Toxicants

3CdCl2/8H2O 0, 5, 10, 20, 30, 50 uM), CoCl3/6H2O (0, 5, 20, 30, 40, 50, 60, 70, 80, 100, 150, 200 uM), CuCl2/2H2O (0, 0.5, 1, 2, 5, 10, 20 uM), NiCl2/6H2O (0, 5, 7.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 22, 25, 30 uM).

Test design

TWo pH levels (5.0 and 6.8) and three replicates was used for each of the four test metals. 1 ml of test culture (in exponential growth phase) was transferred into 125 ml Erlenmeyer flasks with 49 ml of Mod.HSM (Sueoka's (1960) High Salt Medium was modified Medium). The concentrations of ionic Cd, Co, Cu and Ni in the growth medium were determined using GEOCHEM, a chemical speciation model that accepts EDTA as a ligand. Exposure period 5 days.

Measurements/observations

Cell densities using by a Coulter counter.

Evaluations

The effects of the four metals and two pH levels on algal growth were determined by comparing EC30 values for the two algal strains (EC30 = the concentration of divalent metal ion that reduced algal cell density to 30% below the corresponding cell density in control solution) following the methods of Bruce and Versteeg (1992).

[ref. ID; 7398]

Test system

The axonemal ATPase activity

Strains

Wild-type (C-239) and mutant (oda6 and ida4).

Inhibitors/concentrations

Dimethylsulfoxide, ethylene glycol, glycerol, methanol (0, 5, 10, 15, 20, 25, 30% v/v).

Temperature

25 degrees C.

Test design

Cells were suspended in a deflagellation solution that consisted of 30% glycerol, 50 mM KCl, 5 mM MgSO4, 1 mM EDTA, 0.5 mM 2-mercaptoethanol and 10 mM Tris-maleate-KOH buffer (pH 7.0). Deflagellation was completed after a 10-min incubation in an ice bath. Detached flagella and cell bodies were separated with centrifugation at 1,750 g for 3 min. The spernatant containing flagella was centrifuged at 8,500 g for 1 hr. The flagella were demembranated in a KMEMT solution (50 mM KCl, 5 mM MgSO4, 1 mM EDTA, 0.5 mM 2-mercaptoethanol and 10 mM Tris-maleate-KOH buffer (pH 7.0)) containing 0.05% Triton X-100 for 10 min. Demembranated flagellar axonemes were centrifuged for 20 min at 8,500 g.
Axonemal ATPase activity were measured in reaction mixtures that basically contained 0.1 mM phosphoenolpyruvate and 10 ug/ml pyruvate kinase in a KMEMT solution as well as various test substances. The reactions were started by mixing with small amount of ATP solution to make final concentration 1 mM and terminated by adding trichloroacetic acid to make final concentration 5%. Reaction time 10 min.
Flagellar axonemes were treated with trypsin to enable sliding between outer doublet microtubules. The sliding disintegration of trypsin treated axonemes was induced by adding ATP, and monitored by change in optical absorbance at a wave-length of 350 nm.

Measurements/observations

Liberated inorganic phosphate, tubidity.