A simple and sensitive bioassay for the detection of toxic materials using a unicellular green alga

Environmental Pollution 64 (1990) 87-91

A Simple and Sensitive Bioassay for the Detection of Toxic Materials using a Unicellular Green Alga M. J. Wren & D. McCarroll Department of Pure and Applied Biology, Leeds University, Leeds, LS2 9JT, UK

(Received 14 July 1989; revised version received 22 November 1989; accepted 28 November 1989)

A BS TRA C T A simple assay for phytotoxicity, using small scale cultures of Chlorella vulgaris, is described. Growth after 3 days' incubation in 0"5 cm 3 medium is assessed by DMSO-acetone extraction and chlorophyll determination. The method is sensitive enough to detect phytotoxicity in small samples of material and this is illustrated by the inhibitory effects of microgram additions of copper, cadmium and zinc.

INTRODUCTION Recent investigations into the cause of phytotoxicity in controlled environments required a method of detecting toxicity in very small samples of material. Preliminary work indicated that seedlings of higher plants were unlikely to be sufficiently sensitive and we therefore experimented with the unicellular green alga Chlorella vulgaris. The assay which is described together with sample results, takes 3 days, is simple to perform and requires a minimum of apparatus.

MATERIAL A N D METHODS Stock cultures of Chlorella vulgaris are maintained in Bold's basal medium (Stein, 1973), incubated at 2 5 ° C constant temperature and 16-h photoperiod (100pmol m -2 s-1 over 400-700nm, supplied by warm white fluorescent 87 Environ. Pollut. 0269-7491/90/$03"50 © 1990 Elsevier Science Publishers Ltd, England. Printed in ~rent Rritnin

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tubes). Each culture is aerated vigorously by passing air through a Whatman In-Line filter and a sintered glass sparge. Growth is rapid and inoculum is prepared from 7 to 21 day old cultures. The assay is carried out in small vials, 6 cm long, made by cutting Pasteur pipettes at positions 5 cm and 11 cm from the wider end and sealing the narrow end in a bunsen flame. These are used once and then discarded. The vials are supported in a transparent rack (drilled with 7 m m diameter holes at 2-5 cm centres), above a sheet of white expanded polystyrene. By pushing the pointed ends into the polystyrene, the vials are held firmly in position. Aeration of the vial contents is carried out through Pasteur pipettes (short type) connected by small plastic regulators of the type produced for use in aquaria. Air (supplied by a Hy-Flo, Model C pump), is first bubbled through a 1-1itre flask partly filled with distilled water. This has the dual effect of humidifying the air and equalising the pressure to four aeration lines. Each line contains one regulator as a bleed valve and 12 or 13 outlets (each with its own regulator) connected in series. Materials under test are placed in the vials and culture medium is added to produce a volume of 400/~1 single strength Bold's medium containing 200 p p m Dow-Corning anti:foam RD emulsion. This concentration of antifoam emulsion prevents the build-up of bubbles which had caused problems in early trials, whilst having no effect on the rate of growth of the alga. Each vial is inoculated by adding 100 #1 of Chlorella inoculum, prepared by diluting a portion of the stock culture with Bold's medium to give a standard absorbance reading. (We use an arbitrary reading of 1.5 at 652 nm using a Pye-Unicam SP-8-400 spectrophotometer, equivalent to 5"0 #g cm - 3 chlorophyll). The air flow is adjusted to give 1-2 bubbles per second and the vials are incubated in continuous light (100 #mol m - 2 s- 1 supplied by warm white fluorescent tubes), a t c o n s t a n t temperature (24-26°C) for 3 days. Growth of the alga is assessed by the extraction and determination of chlorophyll, using a mixture of dimethylsulphoxide (DMSO) and acetone (Shoaf & Lium, 1976). Vial contents and washings from vials and bubblers are transferred to 1.5 cm 3 Eppendorf tubes and centrifuged for 5 min in a Microspin 24 (Sorvall). The supernatant.is removed carefully with a syringe and discarded before resuspending the pellet in 0.5 cm 3 DMSO. Each vial is left in darkness for a minimum of 10 min, or until all the other vials have been dealt with. Ninety per cent acetone (0.5 cm 3) is then added and the vials are again left in darkness for at least 10 min. After a second centrifugation to remove cell debris, the clear solution is transferred to a glass cuvette and absorbance at 645nm and 663nm is read against a reference cuvette containing equal volumes of D M S O and 90% acetone. Total chlorophyll is calculated as 20-2 A645 + 8.02 A663/2g chlorophyll per vial (Harborne, 1973).

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RESULTS The assay method was tested by examining the effects o f copper, cadmium and zinc supplied individually over a range o f concentrations (5-25 mg litre- 1 Cu + + ; 20-100 mg litre- 1 Cd ÷ ÷ and Zn ÷ ÷). A q u e o u s solutions o f C u C I 2 . 2 H 2 0 , Cd(NO3) 2 . 4 H 2 0 and Z n S O 4 . 7 H 2 0 were placed in the vials prior to inoculation with Chlorella to produce the results shown in Fig. l(a-c). Each c o m p o u n d caused a sharp reduction in algal growth over a narrow range and median inhibitory concentrations were estimated as 7.0 mg litre- 1 Cu, 50"0 mg litre- 1 Cd and 65 mg litre- 1 Zn. 8

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(c) Fig. 1. Effectsof(a) copper, (b) cadmium and (c) zinc on the chlorophyll content (/tg per vial) of Chlorella trulgaris after 3 days' growth in 0.5 cm3 Boid's medium. Points represent means of three replicates _SE. DISCUSSION Algae have been used previously for the assessment of water quality and pollution, in ecotoxicological impact studies and in the development of antifouling treatments for ships and marine structures. (Van Coillie et al.,

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M. J. Wren, D. McCarroll

1983; French & Evans, 1988). Existing methods have been reviewed by Whitton; Walsh and Merrill; and Elnabarawy and Welter, in a useful discussion of algae as ecological indicators (Shubert, 1984). The assay described here was developed specifically to examine air samples from plant growth rooms and it has proved capable of detecting phytotoxicity in very small samples of material collected by passing air through adsorbent filters. Details of this work will be published shortly. The heavy metals copper, cadmium and zinc were used to lillustrate the potential usefulness of the assay, which differs from most of the published methods in its scale. Other workers have used larger volumes or longer culture periods, e.g. Joubert (1983) grew Selenastrum in 25 cm 3 cultures for 8 days. By culturing in only 0"5 cm 3 Bold's medium for 3 days, the Chlorella assay was able to detect less than 3-5 #g Cu, 25/~g Cd and 32.5/~g Zn. Other advantages of the assay are that it is simple to perform, requires no elaborate apparatus and is reliable. Chlorella grows rapidly in aerated culture and no special precautions are used to maintain sterility during the assay period, although the stock culture is grown axenically and the culture medium is autoclaved prior to dispensing into previously heated vials. A single treatment with DMSO-acetone mixture is sufficient to extract all chlorophyll from the cells and the use of triplicates allows standard errors to be calculated. Although the method described is adequate for our purpose, we suggest that certain modifications may improve its sensitivity. Figure 2 shows that omitting the chelating agent ethylenediaminetetraacetic acid (EDTA) and reducing the micronutrient levels in the assay medium caused the median inhibitory concentration of copper to decrease from 7 mg litre ~ in Bold's to l mglitre -1 in Foster's medium (Foster, 1977). This is close to the concentration range of 0.2-0.4 mg litre- ~ Cu which Butler et al. (1980) found increased the lag phase in Chlorella cultures. Other genera may be more sensitive than Chlorella, which Palmer (1969), described as one of the five most pollution tolerant fresh-water algae. Reports that exposure to 8 #g 10

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litre- 1 Cd reduced growth in Selenastrum capricornutum (Van Coillie et al., 1983), suggest that this organism should be tested under our regime. It is also possible that the 3 days currently required for the assay may be reduced by measurement o f physiological parameters such as chlorophyll fluorescence instead o f chlorophyll content.

REFERENCES Butler, M., Haskew, E. J. & Young, M. M. (1980). Copper tolerance in the green alga Chlorella vulgaris. Plant, Cell and Environ., 3, 119-26. Elnabarawy, M. T. & Welter, A. N. (1984). Utilisation of algal cultures and assays by industry. In Algae as Ecological Indicators. ed. L. E. Shubert, Academic Press, pp. 317 28. Foster, P. L. (1977). Copper exclusion as a mechanism of heavy metal tolerance in a green alga. Nature, 269, 322-3.. French, M. S. & Evans, L. V. (1988). The effects of copper and zinc on growth of the fouling diatoms Amphora and Amphiprora. Biofouling, 1, 3-18. Harborne, J. B. (1973). Phytochemical Methods. Chapman and Hall. Joubert, G. (1983). Detailed method for quantitative toxicity measurements using the green algae Selenastrum capricornutum. In Aquatic Toxicology. ed. J. O. Nriagu. Advances in Environmental Science and Technology, Vol. 13. Wiley. pp. 467-85. Palmer, C. M. (1969). A composite rating of algae tolerating organic pollution. J. Phycol., 5, 78 82. Shoaf, W. T. & Lium, B. W. (1976). Improved extraction of chlorophyll a and b from algae using dimethyl sulfoxide. Limnol. Oceanogr., 21,926-8. Shubert, L. E. (1984). Algae as Ecological Indicators. Academic Press. Stein, J. (1973). Handbook of Phycological Methods. Culture Methods and Growth Measurements. Cambridge University Press. Van Coillie, R., Couture, P. & Visser, S. A. (1983). Use of algae in aquatic ecotoxicology. In Aquatic Toxicology. ed. J. O. Nriago. Advances in Environmental Science and Technology, Vol. 13. Wiley. pp. 487-501. Walsh, G. E. & Merrill, R. G. (1984). Algal bioassays of industrial and energy process effluents. In Algae as Ecological Indicators. ed. L. E. Shubert, Academic Press, pp. 329-60. Whitton, B. A. (1984). Algae as monitors of heavy metals in freshwaters. In Algae as Ecological Indicators. ed. L. E. Shubert, Academic Press, pp. 257-80.