Effect of Acetone on the Toxicity of Four Chemicals to Selenastrum Capricornutum

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Bull. Environ. Contam. Toxicol. (1986) 36:254-259 ™ E e j C n v itraomnim a tnitoanl on n |and Toxicology Effect of Acetone on the Toxicity of Four Chemicals to Selenastrum capricornutum N. Adams, ~ K. H, Goulding, 2 and A. J. Dobbs* 1princes Risborough Laboratory, Princes Risborough, Buckinghamshire and 2School of Natural Sciences, The Hatfield Polytechnic, PO Box 109, Hatfield, Hertfordshire, United Kingdom Most procedures for the assessment of algal growth inhibition allow the use of a wa
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  Bull. Environ. Contam. Toxicol. (1986) 36:254-259 ™ j EnvironmentalContamination|and Toxicology Effect of Acetone on the Toxicity of Four Chemicals to Selenastrum capricornutum N. Adams, ~ K. H, Goulding, 2 and A. J. Dobbs*1princes Risborough Laboratory, Princes Risborough, Buckinghamshire and2School of Natural Sciences, The Hatfield Polytechnic, PO Box 109,Hatfield, Hertfordshire, United Kingdom Most procedures for the assessment of algal growthinhibition allow the use of a water miscible organic solventas a carrier for chemicals sparingly soluble in water (ISO,1984; OECD, 1983). In general the procedures limit thecarrier solvent concentration in the final culture toapproximately 100 mg 1 -I -100 ~i i-I). At this concentrationthe organic solvent would not be expected to have anysignificant effect on the solubility of the test chemical; soit is best described as a carrier solvent, rather thanco-solvent which is sometimes used as an alternative.Herzel and Murty (1984) showed that the water solubility ofdieldrin and nitrofen was unaffected by acetone up to500 mg 1 -t, but that captan was 41 per cent more soluble.They express the view that, on the whole, the effect onsolubility is insignificant. In any toxicity test, however,the important factor is what effect the presence of the solventhas on the toxicity of the test compounds.A small number of carrier solvents have been used in algalgrowth inhibition tests. Hughes and Vilkas (1983) recommendN,N-Dimethyl formamide (DMF) as a solvent because of itsresistance to metabolism by bacteria (which grow in closeassociation with algae in non-axenic cultures) and Lundy et al(1984) used methanol as a carrier solvent when testing theeffect of polychlorinated biphenyls, DDT and dieldrin on mixedcultures of marine diatoms. However, the most commonly usedsolvent in algal toxicity testing is acetone. Stratton andCorke (1981) studied the effects of atrazine and permethrin ony PRL. Present Address: Water Research Centre,PO Box 16, Henley Road, Medmenham, Marlow, Buckinghamshire,UK. 254  Scenedesmus quadricauda and Chlorella pyrenoidosa in thepresence of a range of acetone concentrations, up to I per cent(10 g i-I). Short term inhibition of photosynthesis(14C02 uptake) by acetone was measured. Althoughacetone alone was not inhibitory to the test algae there was arange of responses in the pesticide-treated cultures.Different acetone levels did not seem to affect permethrintoxicity but affected atrazine toxicity in an unpredictablemanner. Kleppel and Mclaughlin (1980) tested for acetonetoxicity against the marine diatom Skeletonema costatum in astudy of the effects of PCB's on different cell densities.They found 0.1 per cent (-1000 mg 1 -I) acetone had no effect onculture growth but did not study its effects on PCB toxicity.Stratton (1984) used a method developed by Stratton et al(1982) for fungi to determine that acetone with a finalconcentration of 0.1 per cent in culture could be used as aCarrier solvent in algal toxicity tests. He states that thislevel does not give inhibition values significantly different(p = 0.95) from the inhibition calculated for acetone-freecultures.Although there have been studies of the effects of carriersolvents themselves on the growth of algae, there has beenlittle work on the effects of carrier solvent on the toxicityto algae of chemicals being tested.The study reported here investigates the effects of acetone at100 mg 1 -I (the recommended final concentration in the OECDguidelines) on the toxic values obtained for chemicals ofdifferent aqueous solubilities. Aniline and tebuthiuron(readily soluble), the poorly soluble butyl ester of 2,4dichlorophenoxy acetic acid (2,4-D BE), and almost insoluble1,1,1-trichloro-2,2-di(chloro- phenyl) ethane (DDT). All weretested against Selenastrum capricornutum using a standard testmethod. The freely soluble compounds were chosen toinvestigate acetone effects other than solubilization of thechemicals by the carrier solvent. For example Parasher et al(1978) and Weinberger et al (1983) suggest that solventsinfluence the uptake of chemicals by algae, possibly byinteracting with cell membranes.MATERIALS AND METHODSThe method used was that described as the 'PRL' methodpreviously (Adams and Dobbs, 1984), modified to cope with theaddition of test chemicals with and without acetone.Stock solutions of aniline and tebuthiuron were made up insterile particle-free deionised water whilst stock solutions of2,4-D BE and DDT were made up in acetone 24 hours before theiraddition to the culture flasks. The concentrations of the 255  stock solutions were such that, when 10 ~l aliquots were addedto 100 ml cultures of exponentially growing S. capricornutum,the test chemicals were present at the requiTed treatmentlevel. A second set of DDT solutions was prepared that gavethe required test concentrations when 20 ~i aliquots were addedto 100 ml cultures. In these, therefore, the final acetoneconcentration was -200 mg 1-~; instead of 100 mg i -~, the OECDrecommended amount. Acetone free DDT and 2,4,-D BE acetone-free flasks were prepared by adding 10 U1 aliquots of the stocksolutions to autoclaved, empty 250 ml narrow necked conicalflasks. The flasks were stored in an incubator at 22~ andshaken at 175 rpm for 24 hours while the acetone evaporated.All experiments were carried out in triplicate, the testchemicals being added to 24 hour old cultures containingapproximately 104 cells mi -~ of exponentially growing S.capricornutum. Aniline and tebuthiuron were added in particlefree deionised water, immediately followed by 10 ~l acetone forthe solvent containing replicates. DDT and 2,4-D BE were addedin acetone and their acetone free replicates were prepared bytransferring the cultures, using aseptic technique, to thesterile flasks containlng the compounds prepared 24 hoursearlier. This was termed day O. The cultures were incubatedas above for 6 days and the cell count for each Clask wasrecorded every 24 hours, using an Elzone 80XY electronicparticle counter (Particle Data Ltd).The cultures containing 5 mg 1 -I DDT were analysed at the endof the experiment tQ establlsh that there was, in fact, DDTpresent. Firstly the DDT was extracted from the cultures with15 ml and 5 ml of hexane. The hexane samples were combined,made up to 25 ml and stored at 4~ until required for analysis.The cultures were then lysed with a Soniprobe and hexane wasagain used to extract any remaining DDT, though the lysedcultures and hexane did not separate out clearly. All thesamples were analysed by gas chromatography, using a Pye 104(Pye Unicam Ltd) with electron capture detection.RESULTS AND DISCUSSIONUsing the daily replicate cell counts for tebuthiuron, anilineand 2,4,-D BE, the areas under the growth curve over differentculture periods were calculated. The areas for each treatmentlevel were compared to the controls using Dunnett's method(Zar, 1974), as described elsewhere (Adams et al, in press), togive the lowest concentrations tested that caused a significanteffect (p = 0.95) (LCSE). LCSE values with and without acetonefor tebuthiuron and 2,4-D BE (Table I) are almost identical,and the close similarity of growth curves for tebuthiuron(Figures I and 2) support the view that acetone has had noeffect. This suggests the use of acetone as a carrier solventwith a final concentration in culture of 100 UI 1 -Iisacceptable. 256  L11~m[101001/zmi1700‚i00~~o01olo 003 03~~0000 ~ 0.30 10 105  (  14IIrl~_ AgocuedAgocued Figure i The effect of tebuthiuron without acetone on growth of  S. capricornutum.  Mean cell number of 3 replieate flasks is recorded at eaeh treatment level. Figure 2 The effect of tebuthiuron with i00 pg ml-i acetone on the growth of  S. capricornutum.  Mean cell number of 3 replicate flasks is recorded at each treatment level. 
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