Research Articles
Toxicity Testing of HVC
Toxicity Testing of Highly Volatile Chemicals with Green Algae -
A New Assay
Werner Brack, Horst Rottler Chair of Ecological Chemistry and Geochemistry, University of Bayreuth, D-95447 Bayreuth, Germany
Abstract A gas-tight system for toxicity testing of highly volatile chemicals with the green alga Chlamydomonasreinhardtiiwas developed. The procedure permits maintenance of constant and defined concentrations of the tested compounds in the vessels. To ensure sufficient CO2-supply, new bipartite test vessels were used. These vessels allowed spatial separation of a HCO3/CO3 2" buffer used for CO2 supply and the alga culture to avoid growth inhibition due to ionic strength. Severalvolatile chlorinated hydrocarbons have been tested. Their EC10 valueswere severalorders of magnitude lower than those obtained with open test systems.
1
Introduction
Until now, there has been no standardised or widely used method for toxicity testing of volatile chemicals in a growth inhibition bioassay with green algae. A suggestion for such an assay is presented in this investigation. A cell multiplication assay with green algae for volatile compounds must meet two important requirements: 1. It must provide a sufficient supply of CO2 to avoid growth inhibition due to CO2 deficiency. 2. There must be a well-defined, constant concentration of the tested compounds during incubation time. The easiest way to meet the first demand is to use systems allowing free gas exchange with the atmosphere. Such tests are standardised in the OECD Guideline for Testing of Chemicals, 201 (1984), which does not specify a dinstict alga species and in DIN 38412 Part 33 (1989) with Scenedesmus subspicatus. With volatile chemicals (BRINGMANN& KOHN, 1978 and GEYER et al., 1985) the evaporation during the test makes it impossible to define the effective concentrations in the test flasks. If the calculated EC-values are based on the concentrations of stock solutions, the toxicity of the volatile compounds will be highly underestimated. BRINGMANN & KOHN (1978), for example, achieved toxicity thresholds for tetrachloromethane and trichlorethylene beyond the solubility in water. However, even in closed test systems, analytical control of the actual concentration of the test compound is very important because of inevitable losses during the preparation of the test solutions. Several attempts have been undertaken to maintain constant and well-defined concentrations of the tested chemicals. All of them used sealed systems, however with different approaches to achieve a sufficient supply of CO 2. KOHN & PATTAgD (1990) shortened the incubation time from ESPR-Environ. Sci. & Pollut. Res. 1 (4) 223-228 (1994) 9 ecomedpublishers, D-86899 Landsberg,Germany
72 hours, as recommended in DIN 38412 Part 33 (1989), to 48 hours to reduce the CO 2 demand. GALASSl & VIGHI (1981) used 2-L flasks, containing only 100 mL of medium, to provide a head space volume large enough to avoid CO2 deficiency. HERMANet al. (1990) added 0.4 % N a H C O 3 to the medium to ensure CO2 supply. GALASSl& VIGHI(1981) and HERMAN et al. (1990) controlled the acutal concentrations of the test compounds analytically, whereas KOHN & PATTARD (1990) did not. All of these investigations showed a drastically higher sensitivity of green algae to volatile compounds compared to those obtained by BglNGMANN & KOHN (1978) or G E a R et al. (1985). All of the methods are capable of reducing the problem of CO 2 deficiency in the cultures, but our own experiments showed that CO 2 remained the limiting factor for algal growth in such systems. According to NYHOLM & K~LQViST (1989), COz-limited test systems are less sensitive because toxic effects will not be expressed unless potential growth is reduced so far that COz transport to the cells does no longer limit algal growth. Also CHEN'(1989) showed in a theoretical approach that nutrient limitation reduces sensitivity of growth inhibition tests. Further disadvantages are the high space demand for the method of GALASSI & VIGHI (1981) and growth inhibition due to the high ionic strength if N a H C O 3 is added to the medium in concentrations high enough to avoid CO 2 deficiency. Therefore, in this investigation a new, closed system is used, in which a K H C O J K z C O 3 buffer to supply the algae with CO2 is employed, but the buffer is separated from the test medium to avoid growth inhibition due to the ionic strength. This was done by using bipartite culture flasks as described later in detail. Similar vessels have been already used in plant physiology for the culturing of Chenopodium cells (HOSEMANN & BARZ, 1977). To estimate sensitivity of the test organism Chlamydomonas reinhardtii, toxicities of several nonvolatile and low volatile compounds, which have been already tested with Scenedesmus subspicatus by BgrNGMAhrN & KOHN (1978), have been determined. Aside from these non-volatile compounds, highly volatile chlorinated hydrocarbons have been tested with this system. These substances, primarily used as solvents, are produced in large amounts and released to the environment. They have been found in almost every compartment of the environment. Until now, almost no realistic toxicity data achieved with closed test systems have been available for these chemicals. 223
Toxicity Testing of HVC 2
Research Articles
Materials and Methods
2.1
Test chemicals, test organism and culture conditions
The test chemicals, their degree of purity and their origin are shown in Table 1. The cell multiplication inhibition test was performed with the unicellular freshwater green alga Chlamydomonas reinhardtii (strain number 11 - 3 2 a SAG) from the Univer-
sity of Gfttingen, Germany. Precuhures and test cultures were grown in the medium for unicellular algae according to KUHL(1962) ( ~ Table 2). Incubation of all cultures was done in a Orbital Incubator (Gallenkamp). The cultures were shaken permanendy with a frequency of 120 rpm. They were illuminated from above with 130 aE/m2s without lightdark cycle. The photosynthetically effective light was determined with a Quantum Sensor from Licor Inc. The temperature was maintained at 20 + 1~ C.
Table 1: Test chemicals, their degree of purity and their origin
Chemical
Purity
Origin
Chemical
Purity
Origin
Monolinuron
99 %
Riedel de H~en
Tetrachloromethane
p.a.
Merck
1,3-Dinitrobenzene
> 99 %
Fluka
1,1,1-Trichloroethane
> 99 %
Fluka
KCN
p.a.
Merck
Tetrachloroethylene
99 %
Merck
CuSO 4
reinst
Merck
Trichloromethane
nanograde
Promochem
Phthalic acid diethylether
> 98 %
Fluka
1,1-Dichloroethylene
> 99 %
Aldrich
Sodium ]auryl sulfate
99 %
Roth
Trichloroethylene
> 99 %
Merck
Cyclohexanone
> 98 %
Fluka
1,1,2-Trichloroethane Dichloromethane
> 99 % nanograde
Fluka Promochem
Table 2: Medium for unicellular algae according to KUHL (1962)
2.3
Nutrient
To test non-volatile compounds, well-defined stock solutions in distilled water and dilution series were prepared and added to an equal volume of double concentrated test medium. To run tests with volatile compounds, saturated solutions in growth medium were prepared. Defined aliquots of these solutions were added to the medium. The effective concentrations of the compounds in the alga culture were determined using G C / E C D (electron capture detector) analysis of extracts at the end of the assay.
Concen- Nutrient tration
Concentration
ling/L]
[mg/L]
KNO3
1011.1
FeSO4 - 7 H20
6.95
NaH2PO4, H20
621.0
Na2EDTA- 2 H20
9.3
Na2HPO4 . 2 H20
89.0
MnSO4. H20
0.169
MgSO4 97 H20
246.5
ZnSO4. 7 H20
0.287
CaCI2 92 H20
14.7
CuSO4. 5 H20
0.00249
H3BO3
0.061
(NH4)6Mo7024.4 H20
0.01235
2.2
CO2 supply
All assays were performed using bipartite vessels as shown in Fig. 1 (-~ p. 225). The lower flask contained 50 mL of the CO 2 buffer with 2 mol K2CO3/2 tool KHCO3 (35/65), which provided, according to WARBURG & KRIPPAHL(1960), an atmospheric concentration of 1.14 % CO 2. The CO 2 concentration in the gas phase of the vessel was determined using G C / T C D (thermal conductivity detector) according to FRUNZKEet al. (1984). 1.2 % were measured without algal growth. Algal growth reduced the CO 2 concentration to a minimum of 0.4 %. This value is still far above atmospheric concentration. Growth inhibition by CO2 deficiency was not expected and was not observed. The upper part of the vessel contained 30 mL of the growth medium with the algae. Both liquids were interconnected by their gas phase. The whole system was sealed gas-tight against the atmosphere. Sampling for measurement of toxicant concentration was possible without opening the vessel through septa with screw caps ( ~ Fig. 1).
224
2.4
Preparation of test solutions
Performance of the test
The CO z buffer and the contaminated medium were filled into the test vessel at least 15 hours before the assay were started by adding the algal inoculum. Continuously shaken and temperated to 20 ~ C, the system was allowed to equilibrate. The inoculum was prepared from 7 day old precultures at a concentration of 3 9 10 s cells/mE For the determination of the cell density in the preculture, an aliquot was taken and mixed with the same volume of 0.25 % glutaraldehyde solution in water to make the cells immobile. The cells were counted using a microscope and an improved Neubauer hemacytometer. For the test 0.5 mL inoculum was added to 30 mL medium. The cell density in the test cultures amounted to 5 9 103 cells/mL at the beginning of the assays. The tests were run for 72 hours. After this period, the algae, which have multiplicated at least by factor 100 during the 72-h incubation time, were taken for determination of biomass. Additional measurements demonstrated that, in the absence of toxic compounds, the algae showed exponential growth during the entire incubation time. The pH in the medium ranged from 6.5 to 7.5. For aUe chemicals, the concentraESPR-Environ. Sci. & Pollut. Res. I (4) 1994
Research Articles
Toxicity Testing of HVC 2.6
f
Septa x~4thscrewcaps
T m
~wth medium (30 ml) with ae and test chemical
rE ~O
The percent inhibition for each toxicant concentration was determined. EC10 and EC50 values, including 95 % confidence intervals, were calculated using the PROBIT routine of SAS. The effects of sodium lauryl sulphate, which had a stimulating effect in low concentrations, and 1,3-dinitrobenzene did not fit well to the PROBIT model even though the data points showed little scattering. For these two compounds, EC values were calculated by linear extrapolation between two measured values. 2.7
E ox
l
32 Buffer (50 ml)
--Ir
6.3 cm
!
Fig. 1: Bipartitetest vesselfor toxicitytestingof highlyvolatilechemicals with green algae
tions were tested in duplicate. Each test series contained three controls without toxicant and two controls with 0.8 mg/L Cu 2. (CuSO4). This concentration reduces algal growth to 50 % and is used to check normal sensitivity of the organisms.
2.5
Determination of the biomass
The method selected to determine the biomass was fluorometric measurement of total chlorophyll, which was extracted with boiling ethanol. There was a good correlation (r > 0.99) between fluorometric measurements and cell counts. Aliquots were taken from each test culture in duplicate, mixed with an equal volume of 0.25 % glutaraldehyde solution in brown glass centrifuge tubes and centrifuged 30 min at 3 000 rpm. The supernatant was removed via a pipette connected to a suction pump. Defined volumes of boiling ethanol were pipetted to the cells. The centrifuge tubes were immediately closed with screw caps, shaken and extracted in the dark for about 18 hours. The extracted chlorophyll was determined fluorometrically using a PAM (pulse amplitude modulation) Fluorometer (Walz). The excitation was done using a red light LED (light emitting diode) (Type UBSR, Stanley) with an emission maximum of 650 nm. Radiation with a wavelength longer than 690 nm was quantitatively removed by a short pass filter (DT Cyan). The fuorescence radiation was detected by a PIN photodiode (Type S 1723, Hamatsu), protected from reflected excitation radiation from wavelengths shorter than 690 nm by a longpass filter (Schott RG 9). Deviations between the duplicates were -< 2 %. ESPR-Environ. Sci. & Vollut. Res. 1 (4) 1994
Statistics
Analysis of volatile chlorinated hydrocarbons
The concentrations of the volatile compounds were determined by GC/ECD after liquid-liquid microextraction. Defined volumes (10 or 100 ilL) were taken in duplicate from the test cultures with a gas-tight syringe through the septa and injected into 2 mL vials containing i mL hexane with trichlorobromomethane as internal standard. The vials were shaken in a centrifugal manner by using a Vortex-mixer. The hexane supernatant was analysed using a Hewlett Packard gas chromatograph (HP 5890) equipped with an ECD. A 30 m capillary column (VOCOL, wide bore, 0.53 mm i.d., 3/~m film thickness, Supelco) and nitrogen carrier gas with a flow rate of 5.9 mL/min through the column were used. The injection was done in splitless mode. The temperature program was selected for each compound. This rapid analytical method was used in to minimize evaporation losses during the extraction process. Further cleaning of the samples was not necessary. Measurements at the beginning and at the end of the assays showed no significant differences in chemical concentrations. Therefore, samples for analysis were taken at the end of the assay in order to avoid leaks in the septa, which could allow evaporation during the test period. Deviations between the duplicates, extracted from the same test culture were less than 5 %. To estimate recovery of this analytical method, 20 mL headspace vials were filled completely with water or alga suspension. The vials were sealed gas-tight with septa. Gravimetrically defined amounts of the volatile chlorinated hydrocarbons were injected via syringe through the septa into the liquids and dissolved. From these solutions samples were taken and extracted as explained above. Recovery of the method amounted to 90 + 5 % and was independent from cell density.
3
Results and D i s c u s s i o n
Dose-response plots of the tested non-volatile and lowvolatile chemicals are presented in Fig. 2. (~ p. 226). 72-h EC10 and 72-h EC50, which are the effective concentration of a chemical by which algal growth is reduced by 10 % or 50 % compared to a control in 72 h, respectively, and the 95 % confidence intervals of these values are shown in Table 3 (~ p. 226). There was good correspondence between the PROBIT model and the measurements for most of the compounds. The 95 % confidence intervals were small compared to those reported in literature (for example, COWGILL et al. 1989).
225
Toxicity Testing of HVC
Research Articles
100
90
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80 70
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CHEMICAL CONCENTRATION [mg/L] Fig. 2: Dose-response plots of non-volatile and low-volatile chemicals. The lines show the plots calculated with PROBIT, the spots represent the measured values. Tested compounds: 1 monolinuron, 2 1,3-dinitrobenzene, 3 KCN, 4 CuSO 4. 5 phthalic acid diethyl ether, 6 sodium lauryl sulphate, 7 cyclohexanone Table 3 : 7 2 h-EC10 and 72 h-EC50 values of non-volatile and low-volatile chemicals with 95 % confidence intervals No.
Chemical
[mg/L]
95 % Conf [mg/L]
72-h EClO
72-h EC50 [mg/L]
95 % Conf [mg/L]
1
Monolinuron
0.0139
0.0116 - 0.0162
0.0431
0.0386 - 0.0483
2
1,3-Dinitrobenzene
0.100
4.d.a)
0,220
N,d.
KCN (as CN*)
0.158
0.138-0.176
0.331
0.309-0.354
4
CuSO+ (as Cu2+)
0.642
0.611 - 0.667
0.800
0.777- 0.826
5
Phthalic acid diethylether
1.02
0.736-1.319
8.24
5.29 - 7.50
6
Sodium lauryl sulphate
14.8
~l.d.
18.8
N.d.
7
Cyclohexanone
3.56
0.40 - 7.93
32.9
17.7 - 85.6
") N.d.: Not determined
In Table 4, the 72-h EC10 values for non-volatile and lowvolatile compounds, which are equivalent to the toxicity thresholds, the lowest concentrations with statistically significant effects, in the presented work with Chlamydomonas reinhardtii, are compared with 192-h toxicity thresholds (TT) of BR1NGMANN & KOHN (1978), achieved with Scenedesmus subspicatus. Except for sodium lauryl sulphate and KCN, all tested compounds showed a higher toxicity in the presented bioassay. The sensitivity for CuSO4 is slightly higher than in the investigation of BRrNG~tarCN & K0r~q (1978). The 72-h EC10 values for the other non-ionic organic compounds tested were about one order of magnitude, in the case of cyclohexanone even two orders of magnitude, smaller than toxicity thresholds of BRINGMANN & KOHN(1978). The comparison of EC10 and TT values obtained after different incubation times by BR2qGMA~rN& KOHN (1978) and KOH~q& PATTA~ (1990) shows that for most of low volatile chemicals different incubation times are of minor importance for toxicity data.
226
Table 4: Comparison of the results with non-volatile compounds with BRINGMaNN & KSHN (1978). a) This investigation, b) Ba~qGMANN & KOHN (1978) 192-h TT: Toxicity Threshold after a incubation time of 192 h
[mg/L]
192-h T T b [mg/L]
Monolinuron
0.0139
0.13
2
1,3-Dinitrobenzene
0.100
0.70
3
KCN (as CN*)
0.158
0.03
$
CuSO 4 (as CU 2+)
0.642
1.1
5
Phthalic acid diethyl ester 1.02
10
3
Sodium lauryl sulfate
14.8
0,02
T
Cyclohexanone
3.56
370
No.
Chemical
72-h E C I O a
ESPR-Environ. Sci. & Pollut. Res. 1 (4) 1994
Research Articles
Toxicity Testing of HVC
Hence, Cblamydomonas reinhardtii, which has already been used as test organism (ERNST et al. 1983; LZEBE& FOCK; GARVEY et al. 1991) was very sensitive to organic compounds. The test was designed to detect organic pollutants in environmental samples as sensitive as possible. Therefore, this organism was used in stead of the less sensitive Scenedesmus subspicatus, which is recommended in DIN 38412 part 33 (1989). Dose-response plots of highly volatile chlorinated hydrocarbons, presented in Fig. 3, show good agreement between measured values and PROBIT calculations. Therefore, the 72-h EC10 and 72-h EC50 values (-* Table 5) could be determined very precisely, as demonstrated by the small 95 % confidence intervals. Data for volatile chlorinated C1 and C2 hydrocarbons show a higher toxicity for chloromethanes than for chloroethylenes with the same number of chlorine atoms. Higher degrees of chlorination enhance the toxicity of the compounds as reported for chlorophenols by SH[GEO~:Aet al. (1988). This rule is valid for chloromethanes and chloroethylenes except 1,1-dichloroethylene, which is more toxic than expected. Toxicity of chloroethanes depends very much on the substitution pattern of the molecule. Despite the same number of chlorine atoms, 1,1,1-trichloroethane is about 100-fold more toxic than 1,1,2-trichloroethane. The 72-h ECS0 of 1,1,2-trichloroethane is of the same order of magnitude as reported by ADEMA & FrNK (1981) for the most sensitive of the investigated algae species. The authors used an incubation time of 96 h and 5 different algae. They obtained 96-h EC50 values from 260 mg/1. The effective concentration was controlled analytically. The 72-h EC10 values of highly volatile compounds in the dosed test system with Chlamydomonas reinhardtii are several orders of magnitude lower than EC10 values or toxicity thresholds described in the literature in which open systems and Scenedesmus subspicatus were used (-" Table 6, p. 228).
Table 5 : 7 2 h-EC10 and 72 h-EC50 values of highly volatile chemicals with 95 % confidence intervals No.
Chemical
72-h EC10 95 % Conf [mg/L1 [mg/L]
8 Tetrachloromethane
72-h EC50 95%Conf [mg/Ll
[mg/L]
0.0717
0.0572-0.0864
0.246
0.217 - 0.278
0.213
0.133-0.288
0.536
0.418 - 0.673
10 Tetrachloroethylene
1.77
1.30-2.19
3.64
3.10-4.18
11 Trichloromethane
3.61
2.55-4.72
13.3
11.00 - 15.77
3.94
2.44-5.15
9.12
7.42-11.3
12.3
9.76 - 14.3
36.5
35.1 - 38.2
26.3
2 1 . 0 - 30.4
57.0
54.0 - 60.6
115
79.1-146
242
2 0 2 - 286
9
1,1,1-Trichloroethane
12
1,1-Dichloroethylene
13 Trichloroethylene 14
1,1,2-Trichlomethane
15 Dichloromethane
However, the 48-h EC10 of trichloromethane obtained with dosed vessels by KOrrN & PATTARD(1990) was also about 100-fold higher than the 72-h EC10 presented in this work. Possible reasons are higher sensitivity of the test algae, CO/-limitation of growth and not quantified losses during the preparation of the test solutions by KOHN & PATTARD (1990), who did not analytically determine the effective concentration. Our own experiments showed that significant losses due to evaporation occur during the preparation of dilution series with volatile compounds in water. Without analysis, the concentrations in the test vessels are overestimated. Even though comparison with data from BRINGMANN 8(: KOHN (1978), GEYER et al. (1985) and KOHN & PATTARD
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1,1,1-Trichloroethane
0.213
430 a, d
10
Tetrachtoroethylene
1.77
-
11
Trichloromethane
3.61
1100 a 3 6 0 c
12
1,1-Dichloroethylene
3.94
240 b
13
Trichloroethylene
12.33
> 1000 a 300 b
14
1,1,2-Trichloroethane
26.3
430 a, d
15
Dichlorornethane
115
-
4
Conclusions
Testing volatile compounds with open test systems leads to an underestimation of toxicity. Data obtained in such a manner as reported in numerous handbooks, for example STEINBERGet al. (1992), RWPEN (1992) should no longer be used for toxicity assessment. The method presented in this paper is an appropriate and sensitive bioassay for volatile and non-volatile organic compounds with controlled and constant concentrations during the test period.
Chlamydomonas reinhardtii showed a higher sensitivity to organic compounds than Scenedesmus subspicatus, which is recommended in DIN 38412 Part 33 (1989). Therefore, it was used in this investigation. However, the test should also be applicable to other alga species such as Scenedesmus subspicatus. The assay is not only applicable for toxicity testing of single compounds. It can be also used for toxicity assessment of volatile fractions of environmental samples such as landfill leacbates or waste water.
Acknowledgements We thank BayrischerForschungsverbundAbfallforschungund Reststoffverwertung (BayFORREST) for their financial support. For technical assistance we thank Natascha Liebig. Regina P6hhacker (Institute of Soil Science, University of Bayreuth) made the GC/TCD for CO2-measurements available to us.
228
5
Literature
ADEMA,D. M. M.; G. J. VINK:A Comparative Study of the Toxicity of 1,1,2-Trichloroethane, Dieldrin, Pentachlorophenol and 3,4-Dichloroaniline for Marine and Fresh Water Organisms, Chemosphere 10, 533 (1981) BRINGMANN, G.; R. KOHN: Grenzwerte der Schadwirkung wassergefiLhrdender Stoffe gegen Blaualgen (Microcystis aeruginosa) und Grtinalgen (Scenedesmus subspicatus) irn Zellvermehrungshemmtest. Vom Wasser 50, 45 (1978) CHEN, C.-Y.: The Effects of LimitingNutrient to Algal Toxicity Assessment: A Theoretical Approach. Tox. Assess. 4, 35 (1989) COWGILL,U. M.; D. P. MILAZZO;B. D. LANDENBERGER:Toxicity of Nine Benchmark Cemicals to Skeletonema costatum, A Marine Diatom. Environm. Toxicol. and Chem. 8,451 (1989) DIN 38412 Part 33: Bestimmung der Hemmwirkung yon Wasserinhaltsstoffenauf Grtinalgen (Scenedesmus-ZellvermehrungsHemmtest) (L 9) (1989) ERNST, R.; C. J. GONZALES;J. ARDITTI:Biological Effects of Surfactants: Part 6 - Effects of Anionic, Non-ionic and Amphoteric Surfactants on a Green Alga (Chlamydomonas). Environm. Pollut. (Series A) 31, 159 (1983) FORST,C.; L. STIEGLITZ,W. ROTH;S. KUHNMUNCH:Determination of Volatile Organic Pollutants in Leachate from Different Landfills. Vom Wasser 72, 295 (1989) FRtrNZKE,K.; W. G. ZUMFT:Rapid, Single Sample Analysis of H z, 02, N2, NO, CO, N20 and CO2 by Isothermical Gas Chromatography: Applicationsto the Study of Bacterial Denetrification.J. Chromatogr. 299, 477 (1984) GALASSI,S.; M. VIGHI:Testing Toxicity of Volatile Substances with Algae. Chemosphere 10, 1123 (1981) GARVEY,J. E.; H. A. OWEN;R. W. WrNNER: Toxicity of Copper of the Green Alga, Chlamydomonas reinhardtii (Chlorophyceae), as Affected by Humic Substancesof Terrestrial and Freshwater Origin. Aquat. Toxicol. 19, 89 (1991) GEYER, H.; I. SCHEUNERT;F. KORTE: The Effects of Organic Environmental Chemicals on the Growth of the Algae Scenedesmus subspicatus: A Contribution to Environmental Biology. Chemosphere 14, 1355 (1985) HERMAN, D. C.; W. E. INNISS;C. I. MAYFIELD:Impact of Volatile Aromatic Hydrocarbons, alone and in Combination, on Growth of the Freshwater Alga Selenastrum capricornutum. Aquat. Toxicol. 18, 87 (1990) HOSEMANN,W.; W. BARZ:Photoautotrophic Growth and Photosynthesis in Cell SuspensionCultures of Chenopodium rubrum. Physiol. Plant. 40, 77 (1977) KUHL,A.: Zur Physiologieder Speicherungkondensierter anorganischer Phosphate in Chlorella. Deutsch. Bot. Ges. (Ed), Beitriige zur Physiologie und Morphologie der Algen. pp 157, Fischer Stuttgart (1962) KOHN, R.; M. PATTARD:Results of the Harmful Effects of Water Pollutants to Green Algae (Scenedesmus subspicatus) in the Cell Multiplication Inhibition Test. Water Res. 24, 31 (1990) LIEBE, B.; H. P. FOCK: Growth and Adaption of the Green Alga Chlamydornonas reinhardtii on Diesel Exhaust Particle Extracts. J. Gen. Microbiol. 138, 973 (1992) NYHOLM,N.; T. KALLQVIST:Methods for Growth Inhibition Toxicity Tests with Freshwater Algae. Environ. Toxicol. Chem. 8,689 (1989) OECD: Alga, Growth Inhibition Test. Guidline for Testing of Chemicals, Organization for Economic Cooperation and Development, Document No. 201, Paris, France (1984) RIPPEN, G.: Handbuch Umweltchemikalien, ecomed Verlag Landsberg/Lech (1992) SHIGEOKA,T.; Y. SATO;Y. TAKEDA;K. YOSH1DA;E. YAMAUCHI:Acute Toxicity of Chlorophenols to Green Algae, Selenastrum capricornuturn and Chlorella vulgaris, and Quantitative Structure-Activity Relationships. Environ. Toxicol. Chem. 7, 847 (1988) STEINBERG,Ch.; J. KERN;G. PITZEN;W. TRAUNSPURGER;H. GEYER: Biomonitoring in Binnengew~isser.ecomed Verlag Landsberg/Lech (1992) WARBURG,O.; G. KRIPPAHL:Weiterentwicldung der manometrischen Methoden (Carbonatgemische). Z. Naturforschg 15 b, 364 (1960) Received:January27, 1994;
Accepted:March 29, 1994
ESPR-Environ. Sci. & Pollut. Res. 1 (4) 1994
Toxicity Testing of HVC
Toxicity Testing of Highly Volatile Chemicals with Green Algae -
A New Assay
Werner Brack, Horst Rottler Chair of Ecological Chemistry and Geochemistry, University of Bayreuth, D-95447 Bayreuth, Germany
Abstract A gas-tight system for toxicity testing of highly volatile chemicals with the green alga Chlamydomonasreinhardtiiwas developed. The procedure permits maintenance of constant and defined concentrations of the tested compounds in the vessels. To ensure sufficient CO2-supply, new bipartite test vessels were used. These vessels allowed spatial separation of a HCO3/CO3 2" buffer used for CO2 supply and the alga culture to avoid growth inhibition due to ionic strength. Severalvolatile chlorinated hydrocarbons have been tested. Their EC10 valueswere severalorders of magnitude lower than those obtained with open test systems.
1
Introduction
Until now, there has been no standardised or widely used method for toxicity testing of volatile chemicals in a growth inhibition bioassay with green algae. A suggestion for such an assay is presented in this investigation. A cell multiplication assay with green algae for volatile compounds must meet two important requirements: 1. It must provide a sufficient supply of CO2 to avoid growth inhibition due to CO2 deficiency. 2. There must be a well-defined, constant concentration of the tested compounds during incubation time. The easiest way to meet the first demand is to use systems allowing free gas exchange with the atmosphere. Such tests are standardised in the OECD Guideline for Testing of Chemicals, 201 (1984), which does not specify a dinstict alga species and in DIN 38412 Part 33 (1989) with Scenedesmus subspicatus. With volatile chemicals (BRINGMANN& KOHN, 1978 and GEYER et al., 1985) the evaporation during the test makes it impossible to define the effective concentrations in the test flasks. If the calculated EC-values are based on the concentrations of stock solutions, the toxicity of the volatile compounds will be highly underestimated. BRINGMANN & KOHN (1978), for example, achieved toxicity thresholds for tetrachloromethane and trichlorethylene beyond the solubility in water. However, even in closed test systems, analytical control of the actual concentration of the test compound is very important because of inevitable losses during the preparation of the test solutions. Several attempts have been undertaken to maintain constant and well-defined concentrations of the tested chemicals. All of them used sealed systems, however with different approaches to achieve a sufficient supply of CO 2. KOHN & PATTAgD (1990) shortened the incubation time from ESPR-Environ. Sci. & Pollut. Res. 1 (4) 223-228 (1994) 9 ecomedpublishers, D-86899 Landsberg,Germany
72 hours, as recommended in DIN 38412 Part 33 (1989), to 48 hours to reduce the CO 2 demand. GALASSl & VIGHI (1981) used 2-L flasks, containing only 100 mL of medium, to provide a head space volume large enough to avoid CO2 deficiency. HERMANet al. (1990) added 0.4 % N a H C O 3 to the medium to ensure CO2 supply. GALASSl& VIGHI(1981) and HERMAN et al. (1990) controlled the acutal concentrations of the test compounds analytically, whereas KOHN & PATTARD (1990) did not. All of these investigations showed a drastically higher sensitivity of green algae to volatile compounds compared to those obtained by BglNGMANN & KOHN (1978) or G E a R et al. (1985). All of the methods are capable of reducing the problem of CO 2 deficiency in the cultures, but our own experiments showed that CO 2 remained the limiting factor for algal growth in such systems. According to NYHOLM & K~LQViST (1989), COz-limited test systems are less sensitive because toxic effects will not be expressed unless potential growth is reduced so far that COz transport to the cells does no longer limit algal growth. Also CHEN'(1989) showed in a theoretical approach that nutrient limitation reduces sensitivity of growth inhibition tests. Further disadvantages are the high space demand for the method of GALASSI & VIGHI (1981) and growth inhibition due to the high ionic strength if N a H C O 3 is added to the medium in concentrations high enough to avoid CO 2 deficiency. Therefore, in this investigation a new, closed system is used, in which a K H C O J K z C O 3 buffer to supply the algae with CO2 is employed, but the buffer is separated from the test medium to avoid growth inhibition due to the ionic strength. This was done by using bipartite culture flasks as described later in detail. Similar vessels have been already used in plant physiology for the culturing of Chenopodium cells (HOSEMANN & BARZ, 1977). To estimate sensitivity of the test organism Chlamydomonas reinhardtii, toxicities of several nonvolatile and low volatile compounds, which have been already tested with Scenedesmus subspicatus by BgrNGMAhrN & KOHN (1978), have been determined. Aside from these non-volatile compounds, highly volatile chlorinated hydrocarbons have been tested with this system. These substances, primarily used as solvents, are produced in large amounts and released to the environment. They have been found in almost every compartment of the environment. Until now, almost no realistic toxicity data achieved with closed test systems have been available for these chemicals. 223
Toxicity Testing of HVC 2
Research Articles
Materials and Methods
2.1
Test chemicals, test organism and culture conditions
The test chemicals, their degree of purity and their origin are shown in Table 1. The cell multiplication inhibition test was performed with the unicellular freshwater green alga Chlamydomonas reinhardtii (strain number 11 - 3 2 a SAG) from the Univer-
sity of Gfttingen, Germany. Precuhures and test cultures were grown in the medium for unicellular algae according to KUHL(1962) ( ~ Table 2). Incubation of all cultures was done in a Orbital Incubator (Gallenkamp). The cultures were shaken permanendy with a frequency of 120 rpm. They were illuminated from above with 130 aE/m2s without lightdark cycle. The photosynthetically effective light was determined with a Quantum Sensor from Licor Inc. The temperature was maintained at 20 + 1~ C.
Table 1: Test chemicals, their degree of purity and their origin
Chemical
Purity
Origin
Chemical
Purity
Origin
Monolinuron
99 %
Riedel de H~en
Tetrachloromethane
p.a.
Merck
1,3-Dinitrobenzene
> 99 %
Fluka
1,1,1-Trichloroethane
> 99 %
Fluka
KCN
p.a.
Merck
Tetrachloroethylene
99 %
Merck
CuSO 4
reinst
Merck
Trichloromethane
nanograde
Promochem
Phthalic acid diethylether
> 98 %
Fluka
1,1-Dichloroethylene
> 99 %
Aldrich
Sodium ]auryl sulfate
99 %
Roth
Trichloroethylene
> 99 %
Merck
Cyclohexanone
> 98 %
Fluka
1,1,2-Trichloroethane Dichloromethane
> 99 % nanograde
Fluka Promochem
Table 2: Medium for unicellular algae according to KUHL (1962)
2.3
Nutrient
To test non-volatile compounds, well-defined stock solutions in distilled water and dilution series were prepared and added to an equal volume of double concentrated test medium. To run tests with volatile compounds, saturated solutions in growth medium were prepared. Defined aliquots of these solutions were added to the medium. The effective concentrations of the compounds in the alga culture were determined using G C / E C D (electron capture detector) analysis of extracts at the end of the assay.
Concen- Nutrient tration
Concentration
ling/L]
[mg/L]
KNO3
1011.1
FeSO4 - 7 H20
6.95
NaH2PO4, H20
621.0
Na2EDTA- 2 H20
9.3
Na2HPO4 . 2 H20
89.0
MnSO4. H20
0.169
MgSO4 97 H20
246.5
ZnSO4. 7 H20
0.287
CaCI2 92 H20
14.7
CuSO4. 5 H20
0.00249
H3BO3
0.061
(NH4)6Mo7024.4 H20
0.01235
2.2
CO2 supply
All assays were performed using bipartite vessels as shown in Fig. 1 (-~ p. 225). The lower flask contained 50 mL of the CO 2 buffer with 2 mol K2CO3/2 tool KHCO3 (35/65), which provided, according to WARBURG & KRIPPAHL(1960), an atmospheric concentration of 1.14 % CO 2. The CO 2 concentration in the gas phase of the vessel was determined using G C / T C D (thermal conductivity detector) according to FRUNZKEet al. (1984). 1.2 % were measured without algal growth. Algal growth reduced the CO 2 concentration to a minimum of 0.4 %. This value is still far above atmospheric concentration. Growth inhibition by CO2 deficiency was not expected and was not observed. The upper part of the vessel contained 30 mL of the growth medium with the algae. Both liquids were interconnected by their gas phase. The whole system was sealed gas-tight against the atmosphere. Sampling for measurement of toxicant concentration was possible without opening the vessel through septa with screw caps ( ~ Fig. 1).
224
2.4
Preparation of test solutions
Performance of the test
The CO z buffer and the contaminated medium were filled into the test vessel at least 15 hours before the assay were started by adding the algal inoculum. Continuously shaken and temperated to 20 ~ C, the system was allowed to equilibrate. The inoculum was prepared from 7 day old precultures at a concentration of 3 9 10 s cells/mE For the determination of the cell density in the preculture, an aliquot was taken and mixed with the same volume of 0.25 % glutaraldehyde solution in water to make the cells immobile. The cells were counted using a microscope and an improved Neubauer hemacytometer. For the test 0.5 mL inoculum was added to 30 mL medium. The cell density in the test cultures amounted to 5 9 103 cells/mL at the beginning of the assays. The tests were run for 72 hours. After this period, the algae, which have multiplicated at least by factor 100 during the 72-h incubation time, were taken for determination of biomass. Additional measurements demonstrated that, in the absence of toxic compounds, the algae showed exponential growth during the entire incubation time. The pH in the medium ranged from 6.5 to 7.5. For aUe chemicals, the concentraESPR-Environ. Sci. & Pollut. Res. I (4) 1994
Research Articles
Toxicity Testing of HVC 2.6
f
Septa x~4thscrewcaps
T m
~wth medium (30 ml) with ae and test chemical
rE ~O
The percent inhibition for each toxicant concentration was determined. EC10 and EC50 values, including 95 % confidence intervals, were calculated using the PROBIT routine of SAS. The effects of sodium lauryl sulphate, which had a stimulating effect in low concentrations, and 1,3-dinitrobenzene did not fit well to the PROBIT model even though the data points showed little scattering. For these two compounds, EC values were calculated by linear extrapolation between two measured values. 2.7
E ox
l
32 Buffer (50 ml)
--Ir
6.3 cm
!
Fig. 1: Bipartitetest vesselfor toxicitytestingof highlyvolatilechemicals with green algae
tions were tested in duplicate. Each test series contained three controls without toxicant and two controls with 0.8 mg/L Cu 2. (CuSO4). This concentration reduces algal growth to 50 % and is used to check normal sensitivity of the organisms.
2.5
Determination of the biomass
The method selected to determine the biomass was fluorometric measurement of total chlorophyll, which was extracted with boiling ethanol. There was a good correlation (r > 0.99) between fluorometric measurements and cell counts. Aliquots were taken from each test culture in duplicate, mixed with an equal volume of 0.25 % glutaraldehyde solution in brown glass centrifuge tubes and centrifuged 30 min at 3 000 rpm. The supernatant was removed via a pipette connected to a suction pump. Defined volumes of boiling ethanol were pipetted to the cells. The centrifuge tubes were immediately closed with screw caps, shaken and extracted in the dark for about 18 hours. The extracted chlorophyll was determined fluorometrically using a PAM (pulse amplitude modulation) Fluorometer (Walz). The excitation was done using a red light LED (light emitting diode) (Type UBSR, Stanley) with an emission maximum of 650 nm. Radiation with a wavelength longer than 690 nm was quantitatively removed by a short pass filter (DT Cyan). The fuorescence radiation was detected by a PIN photodiode (Type S 1723, Hamatsu), protected from reflected excitation radiation from wavelengths shorter than 690 nm by a longpass filter (Schott RG 9). Deviations between the duplicates were -< 2 %. ESPR-Environ. Sci. & Vollut. Res. 1 (4) 1994
Statistics
Analysis of volatile chlorinated hydrocarbons
The concentrations of the volatile compounds were determined by GC/ECD after liquid-liquid microextraction. Defined volumes (10 or 100 ilL) were taken in duplicate from the test cultures with a gas-tight syringe through the septa and injected into 2 mL vials containing i mL hexane with trichlorobromomethane as internal standard. The vials were shaken in a centrifugal manner by using a Vortex-mixer. The hexane supernatant was analysed using a Hewlett Packard gas chromatograph (HP 5890) equipped with an ECD. A 30 m capillary column (VOCOL, wide bore, 0.53 mm i.d., 3/~m film thickness, Supelco) and nitrogen carrier gas with a flow rate of 5.9 mL/min through the column were used. The injection was done in splitless mode. The temperature program was selected for each compound. This rapid analytical method was used in to minimize evaporation losses during the extraction process. Further cleaning of the samples was not necessary. Measurements at the beginning and at the end of the assays showed no significant differences in chemical concentrations. Therefore, samples for analysis were taken at the end of the assay in order to avoid leaks in the septa, which could allow evaporation during the test period. Deviations between the duplicates, extracted from the same test culture were less than 5 %. To estimate recovery of this analytical method, 20 mL headspace vials were filled completely with water or alga suspension. The vials were sealed gas-tight with septa. Gravimetrically defined amounts of the volatile chlorinated hydrocarbons were injected via syringe through the septa into the liquids and dissolved. From these solutions samples were taken and extracted as explained above. Recovery of the method amounted to 90 + 5 % and was independent from cell density.
3
Results and D i s c u s s i o n
Dose-response plots of the tested non-volatile and lowvolatile chemicals are presented in Fig. 2. (~ p. 226). 72-h EC10 and 72-h EC50, which are the effective concentration of a chemical by which algal growth is reduced by 10 % or 50 % compared to a control in 72 h, respectively, and the 95 % confidence intervals of these values are shown in Table 3 (~ p. 226). There was good correspondence between the PROBIT model and the measurements for most of the compounds. The 95 % confidence intervals were small compared to those reported in literature (for example, COWGILL et al. 1989).
225
Toxicity Testing of HVC
Research Articles
100
90
d" P ' ~
80 70
~
6o
~
50
Iin ~.
~
;
o
ti i
,'
/
3o 2o fo
:d 2t,;
f4
;
/ '//i
:!
J/
-lO
I
/
/,
/
/
"'A
dr r
6 /
o
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z~
/
.-"..',
i /
~
0.01
?
:
I 1"
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0.1
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100
1000
l "
-,30
CHEMICAL CONCENTRATION [mg/L] Fig. 2: Dose-response plots of non-volatile and low-volatile chemicals. The lines show the plots calculated with PROBIT, the spots represent the measured values. Tested compounds: 1 monolinuron, 2 1,3-dinitrobenzene, 3 KCN, 4 CuSO 4. 5 phthalic acid diethyl ether, 6 sodium lauryl sulphate, 7 cyclohexanone Table 3 : 7 2 h-EC10 and 72 h-EC50 values of non-volatile and low-volatile chemicals with 95 % confidence intervals No.
Chemical
[mg/L]
95 % Conf [mg/L]
72-h EClO
72-h EC50 [mg/L]
95 % Conf [mg/L]
1
Monolinuron
0.0139
0.0116 - 0.0162
0.0431
0.0386 - 0.0483
2
1,3-Dinitrobenzene
0.100
4.d.a)
0,220
N,d.
KCN (as CN*)
0.158
0.138-0.176
0.331
0.309-0.354
4
CuSO+ (as Cu2+)
0.642
0.611 - 0.667
0.800
0.777- 0.826
5
Phthalic acid diethylether
1.02
0.736-1.319
8.24
5.29 - 7.50
6
Sodium lauryl sulphate
14.8
~l.d.
18.8
N.d.
7
Cyclohexanone
3.56
0.40 - 7.93
32.9
17.7 - 85.6
") N.d.: Not determined
In Table 4, the 72-h EC10 values for non-volatile and lowvolatile compounds, which are equivalent to the toxicity thresholds, the lowest concentrations with statistically significant effects, in the presented work with Chlamydomonas reinhardtii, are compared with 192-h toxicity thresholds (TT) of BR1NGMANN & KOHN (1978), achieved with Scenedesmus subspicatus. Except for sodium lauryl sulphate and KCN, all tested compounds showed a higher toxicity in the presented bioassay. The sensitivity for CuSO4 is slightly higher than in the investigation of BRrNG~tarCN & K0r~q (1978). The 72-h EC10 values for the other non-ionic organic compounds tested were about one order of magnitude, in the case of cyclohexanone even two orders of magnitude, smaller than toxicity thresholds of BRINGMANN & KOHN(1978). The comparison of EC10 and TT values obtained after different incubation times by BR2qGMA~rN& KOHN (1978) and KOH~q& PATTA~ (1990) shows that for most of low volatile chemicals different incubation times are of minor importance for toxicity data.
226
Table 4: Comparison of the results with non-volatile compounds with BRINGMaNN & KSHN (1978). a) This investigation, b) Ba~qGMANN & KOHN (1978) 192-h TT: Toxicity Threshold after a incubation time of 192 h
[mg/L]
192-h T T b [mg/L]
Monolinuron
0.0139
0.13
2
1,3-Dinitrobenzene
0.100
0.70
3
KCN (as CN*)
0.158
0.03
$
CuSO 4 (as CU 2+)
0.642
1.1
5
Phthalic acid diethyl ester 1.02
10
3
Sodium lauryl sulfate
14.8
0,02
T
Cyclohexanone
3.56
370
No.
Chemical
72-h E C I O a
ESPR-Environ. Sci. & Pollut. Res. 1 (4) 1994
Research Articles
Toxicity Testing of HVC
Hence, Cblamydomonas reinhardtii, which has already been used as test organism (ERNST et al. 1983; LZEBE& FOCK; GARVEY et al. 1991) was very sensitive to organic compounds. The test was designed to detect organic pollutants in environmental samples as sensitive as possible. Therefore, this organism was used in stead of the less sensitive Scenedesmus subspicatus, which is recommended in DIN 38412 part 33 (1989). Dose-response plots of highly volatile chlorinated hydrocarbons, presented in Fig. 3, show good agreement between measured values and PROBIT calculations. Therefore, the 72-h EC10 and 72-h EC50 values (-* Table 5) could be determined very precisely, as demonstrated by the small 95 % confidence intervals. Data for volatile chlorinated C1 and C2 hydrocarbons show a higher toxicity for chloromethanes than for chloroethylenes with the same number of chlorine atoms. Higher degrees of chlorination enhance the toxicity of the compounds as reported for chlorophenols by SH[GEO~:Aet al. (1988). This rule is valid for chloromethanes and chloroethylenes except 1,1-dichloroethylene, which is more toxic than expected. Toxicity of chloroethanes depends very much on the substitution pattern of the molecule. Despite the same number of chlorine atoms, 1,1,1-trichloroethane is about 100-fold more toxic than 1,1,2-trichloroethane. The 72-h ECS0 of 1,1,2-trichloroethane is of the same order of magnitude as reported by ADEMA & FrNK (1981) for the most sensitive of the investigated algae species. The authors used an incubation time of 96 h and 5 different algae. They obtained 96-h EC50 values from 260 mg/1. The effective concentration was controlled analytically. The 72-h EC10 values of highly volatile compounds in the dosed test system with Chlamydomonas reinhardtii are several orders of magnitude lower than EC10 values or toxicity thresholds described in the literature in which open systems and Scenedesmus subspicatus were used (-" Table 6, p. 228).
Table 5 : 7 2 h-EC10 and 72 h-EC50 values of highly volatile chemicals with 95 % confidence intervals No.
Chemical
72-h EC10 95 % Conf [mg/L1 [mg/L]
8 Tetrachloromethane
72-h EC50 95%Conf [mg/Ll
[mg/L]
0.0717
0.0572-0.0864
0.246
0.217 - 0.278
0.213
0.133-0.288
0.536
0.418 - 0.673
10 Tetrachloroethylene
1.77
1.30-2.19
3.64
3.10-4.18
11 Trichloromethane
3.61
2.55-4.72
13.3
11.00 - 15.77
3.94
2.44-5.15
9.12
7.42-11.3
12.3
9.76 - 14.3
36.5
35.1 - 38.2
26.3
2 1 . 0 - 30.4
57.0
54.0 - 60.6
115
79.1-146
242
2 0 2 - 286
9
1,1,1-Trichloroethane
12
1,1-Dichloroethylene
13 Trichloroethylene 14
1,1,2-Trichlomethane
15 Dichloromethane
However, the 48-h EC10 of trichloromethane obtained with dosed vessels by KOrrN & PATTARD(1990) was also about 100-fold higher than the 72-h EC10 presented in this work. Possible reasons are higher sensitivity of the test algae, CO/-limitation of growth and not quantified losses during the preparation of the test solutions by KOHN & PATTARD (1990), who did not analytically determine the effective concentration. Our own experiments showed that significant losses due to evaporation occur during the preparation of dilution series with volatile compounds in water. Without analysis, the concentrations in the test vessels are overestimated. Even though comparison with data from BRINGMANN 8(: KOHN (1978), GEYER et al. (1985) and KOHN & PATTARD
100 /
90
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80
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o..'," :/
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600 a
9
1,1,1-Trichloroethane
0.213
430 a, d
10
Tetrachtoroethylene
1.77
-
11
Trichloromethane
3.61
1100 a 3 6 0 c
12
1,1-Dichloroethylene
3.94
240 b
13
Trichloroethylene
12.33
> 1000 a 300 b
14
1,1,2-Trichloroethane
26.3
430 a, d
15
Dichlorornethane
115
-
4
Conclusions
Testing volatile compounds with open test systems leads to an underestimation of toxicity. Data obtained in such a manner as reported in numerous handbooks, for example STEINBERGet al. (1992), RWPEN (1992) should no longer be used for toxicity assessment. The method presented in this paper is an appropriate and sensitive bioassay for volatile and non-volatile organic compounds with controlled and constant concentrations during the test period.
Chlamydomonas reinhardtii showed a higher sensitivity to organic compounds than Scenedesmus subspicatus, which is recommended in DIN 38412 Part 33 (1989). Therefore, it was used in this investigation. However, the test should also be applicable to other alga species such as Scenedesmus subspicatus. The assay is not only applicable for toxicity testing of single compounds. It can be also used for toxicity assessment of volatile fractions of environmental samples such as landfill leacbates or waste water.
Acknowledgements We thank BayrischerForschungsverbundAbfallforschungund Reststoffverwertung (BayFORREST) for their financial support. For technical assistance we thank Natascha Liebig. Regina P6hhacker (Institute of Soil Science, University of Bayreuth) made the GC/TCD for CO2-measurements available to us.
228
5
Literature
ADEMA,D. M. M.; G. J. VINK:A Comparative Study of the Toxicity of 1,1,2-Trichloroethane, Dieldrin, Pentachlorophenol and 3,4-Dichloroaniline for Marine and Fresh Water Organisms, Chemosphere 10, 533 (1981) BRINGMANN, G.; R. KOHN: Grenzwerte der Schadwirkung wassergefiLhrdender Stoffe gegen Blaualgen (Microcystis aeruginosa) und Grtinalgen (Scenedesmus subspicatus) irn Zellvermehrungshemmtest. Vom Wasser 50, 45 (1978) CHEN, C.-Y.: The Effects of LimitingNutrient to Algal Toxicity Assessment: A Theoretical Approach. Tox. Assess. 4, 35 (1989) COWGILL,U. M.; D. P. MILAZZO;B. D. LANDENBERGER:Toxicity of Nine Benchmark Cemicals to Skeletonema costatum, A Marine Diatom. Environm. Toxicol. and Chem. 8,451 (1989) DIN 38412 Part 33: Bestimmung der Hemmwirkung yon Wasserinhaltsstoffenauf Grtinalgen (Scenedesmus-ZellvermehrungsHemmtest) (L 9) (1989) ERNST, R.; C. J. GONZALES;J. ARDITTI:Biological Effects of Surfactants: Part 6 - Effects of Anionic, Non-ionic and Amphoteric Surfactants on a Green Alga (Chlamydomonas). Environm. Pollut. (Series A) 31, 159 (1983) FORST,C.; L. STIEGLITZ,W. ROTH;S. KUHNMUNCH:Determination of Volatile Organic Pollutants in Leachate from Different Landfills. Vom Wasser 72, 295 (1989) FRtrNZKE,K.; W. G. ZUMFT:Rapid, Single Sample Analysis of H z, 02, N2, NO, CO, N20 and CO2 by Isothermical Gas Chromatography: Applicationsto the Study of Bacterial Denetrification.J. Chromatogr. 299, 477 (1984) GALASSI,S.; M. VIGHI:Testing Toxicity of Volatile Substances with Algae. Chemosphere 10, 1123 (1981) GARVEY,J. E.; H. A. OWEN;R. W. WrNNER: Toxicity of Copper of the Green Alga, Chlamydomonas reinhardtii (Chlorophyceae), as Affected by Humic Substancesof Terrestrial and Freshwater Origin. Aquat. Toxicol. 19, 89 (1991) GEYER, H.; I. SCHEUNERT;F. KORTE: The Effects of Organic Environmental Chemicals on the Growth of the Algae Scenedesmus subspicatus: A Contribution to Environmental Biology. Chemosphere 14, 1355 (1985) HERMAN, D. C.; W. E. INNISS;C. I. MAYFIELD:Impact of Volatile Aromatic Hydrocarbons, alone and in Combination, on Growth of the Freshwater Alga Selenastrum capricornutum. Aquat. Toxicol. 18, 87 (1990) HOSEMANN,W.; W. BARZ:Photoautotrophic Growth and Photosynthesis in Cell SuspensionCultures of Chenopodium rubrum. Physiol. Plant. 40, 77 (1977) KUHL,A.: Zur Physiologieder Speicherungkondensierter anorganischer Phosphate in Chlorella. Deutsch. Bot. Ges. (Ed), Beitriige zur Physiologie und Morphologie der Algen. pp 157, Fischer Stuttgart (1962) KOHN, R.; M. PATTARD:Results of the Harmful Effects of Water Pollutants to Green Algae (Scenedesmus subspicatus) in the Cell Multiplication Inhibition Test. Water Res. 24, 31 (1990) LIEBE, B.; H. P. FOCK: Growth and Adaption of the Green Alga Chlamydornonas reinhardtii on Diesel Exhaust Particle Extracts. J. Gen. Microbiol. 138, 973 (1992) NYHOLM,N.; T. KALLQVIST:Methods for Growth Inhibition Toxicity Tests with Freshwater Algae. Environ. Toxicol. Chem. 8,689 (1989) OECD: Alga, Growth Inhibition Test. Guidline for Testing of Chemicals, Organization for Economic Cooperation and Development, Document No. 201, Paris, France (1984) RIPPEN, G.: Handbuch Umweltchemikalien, ecomed Verlag Landsberg/Lech (1992) SHIGEOKA,T.; Y. SATO;Y. TAKEDA;K. YOSH1DA;E. YAMAUCHI:Acute Toxicity of Chlorophenols to Green Algae, Selenastrum capricornuturn and Chlorella vulgaris, and Quantitative Structure-Activity Relationships. Environ. Toxicol. Chem. 7, 847 (1988) STEINBERG,Ch.; J. KERN;G. PITZEN;W. TRAUNSPURGER;H. GEYER: Biomonitoring in Binnengew~isser.ecomed Verlag Landsberg/Lech (1992) WARBURG,O.; G. KRIPPAHL:Weiterentwicldung der manometrischen Methoden (Carbonatgemische). Z. Naturforschg 15 b, 364 (1960) Received:January27, 1994;
Accepted:March 29, 1994
ESPR-Environ. Sci. & Pollut. Res. 1 (4) 1994
