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Algal assay evaluation of trace contaminants in surface water using the nonionic surfactant, triton X-100
Aquatic Toxicology, 6 (1985) 115-131 Elsevier
115
AQT 00146 ALGAL
ASSAY EVALUATION
FACE WATER
OF TRACE
USING THE NONIONIC
CONTAMINANTS
SURFACTANT,
IN SUR-
TRITON
X-100
S.L. WONG
Ontario Ministry o f the Environment, Aquatic Contaminants Section, Water Resources Branch, P.O. Box 213, Rexdale, Ontario M g w 5LI, Canada (Received 2 April 1984; revised version received and accepted 4 December 1984)
Triton X-100, a nonionic surfactant, has been found to be capable of neutralizing the toxic effect of selected organic compounds in algal assays using 10o7o Bristol's medium. This nonionic surfactant does not seem to affect specific growth rates of Chlorella cultures when it is added alone to the medium but it accelerates growth inhibition when coupled with metals. By administering a series of increasing Triton concentrations in assays with natural waters, Chlorella growth that was once arrested in the test waters became revitalized and growth was more profuse as the concentration of Triton increased. The results indicate that Triton has the ability to alleviate the toxic stresses of organic mixtures that are contaminating many southern Ontario lakes. The bioavailability of these trace organics such as PCB, DDT, chlordane and dieldrin was confirmed in the study lakewaters from bioaccumulation in fish tissues. Key words: trace organic contaminants; metals; natural waters; algal assay; surfactant; Chlorella fusca
INTRODUCTION
Many lakewaters in southern Ontario were found to be incapable of supporting algal growth in assays when they were used as culture media (Wong, 1984). After repeated attempts failed to initiate Chlorella growth with both nutrients and the chelator, NazEDTA, it appeared that the inhibition of algal growth was much more than just the deficiency of nutrients or the toxicity of metals. Organic trace contaminants were therefore suspected. The following study was undertaken to test the hypothesis that the inhibition of algal growth in these assays might have been due to stress from organic contaminants prevalent in aquatic environments (Donaldson, 1977; Lunde and Bjorbeth, 1977; Metcalf, 1977; Alfhiem et al., 1978; Zoetman et al., 1980; Cooke and Dennis, 1981). So far, there has not been an appropriate method developed for assessing trace organic contaminants in natural waters despite the many algal toxicity assay techniques available - some are well developed (USEPA, 1978; APHA, 1980; ISO, 1980; OECD, 1981) and some have greatly improved (Payne and Hall, 1978; Joubert, 1980; Blaise et al., 1983; Ferrard et al., 1983). 0166-445X/85/$03.30
© 1985 Elsevier Science Publishers B.V. (Biomedical Division)
116
Apparently, toxicity assay methods developed thus far, are mostly concerned with the management of industrial wastewaters. Since it is essential to know the amount of dilution needed to lower the toxicity of a waste effluent to an acceptable level, the dilution technique, to produce an LCs0 value (lethal concentration), becomes the backbone of conventional approaches. However, surface waters on the other hand, contain numerous trace contaminants, with some 500-700 new organic chemicals entering each year (Metcalf, 1977). The contaminants, though trace in amount, can be highly toxic at times, e.g., dioxin. It is therefore difficult to determine, by employing the dilution technique, how m a n y toxic substances are being 'washed away' or by how much water quality has been changed. Most of all, dilution is not a technique used to single out organic toxicity in water. Therefore, in order to evaluate the organic toxic stress in these waters and to quantify the growth inhibition of algae in the system, an alternative algal toxicity test has been suggested in this paper for the examination of trace contaminants. MATERIALS AND METHODS
Test organism The green, unicellular alga, Chlorellafusca Shihers et Krauses obtained from the C a n a d a Centre for Inland Waters in Burlington, Ontario, was used as the test organism in this study. C. fusca showed high tolerance to metals (Wong and Beaver, 1980), organics (Geyer et al., 1984) and low p H (Hutchinson and Stokes, 1975) in assays. Unlike the universal test alga, Selenastrum capricornutum which is very sensitive to toxicant, Chlorella often achieves sufficient biomass for tissue analysis of contaminants. It is preferred as a test alga in a toxicity study such as this. Chlorella cultures were maintained in 10% Bristol's solution (Starr, 1964) and were used as an inoculum only when the cells reached the logarithmic phase of growth, that is between the 7th and 10th day.
Triton )(-100, a specific test compound Triton X-100 (isooctylphenoxypolyethoxyethanol), a nonionic surface active agent, has the following structural formula:
R"~ ~"O(OCH2CH2)10OH It is biodegradable and water soluble and it contains an average of 10 mol ethylene oxide in the octylphenol series. It emulsifies and solubilizes organic solvent in solution and has been used to remove lipid, hydrocarbon and other organic stains by textile industries (Cruickshank, 1968; Anon., 1981; Lillie et al., 1982).
117
Assays o f natural waters Surface water samples were collected f r o m 13 lakes, reservoirs and bays in southern Ontario in the summer of 1981 and 1982 (Table I). Each sample was separated into two parts on arrival at the laboratory. One part was for routine chemical analyses which included p H , hardness, dissolved organic carbon (DOC), total phosphorus (TP) and total Kjeldahl nitrogen (TKN); the other part was used for algal toxicity tests. Water samples were filtered through a 0.45 #m membrane filter and the Erlenmeyer flasks (250 ml) were acid washed before use. The assay was carried out by dividing a filtered water sample into seven 90-ml portions with duplicates. Ten ml of normal Bristol's solution were added to each portion to provide a 10°70 Bristol's medium so as to eliminate any possible deficiency o f nutrients, especially those from the softwater lakes (hardness<20 mg CaCO3" 1- 1). The first portion was used as a control; the second portion, to which 10 m g . l - 1 Na2EDTA were added, was used for evaluation o f metal toxicity; the 3rd, 4th, 5th, 6th and 7th portions contained Triton X-100 in concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0 m M , respectively. One ml of Chlorella cells was inoculated into each flask to give an initial concentration o f about 8 × 104 cells per ml. The culture flasks were mounted on a platform shaker set at 90 oscillations per min. The cultures were illuminated on a 12:12 light-dark cycle by cool, white fluorescent light at about 8000 lux and were kept at temperatures between 18-20°C. The optical densities of the initial cell concentrations were read at 730 nm with a spectrophotometer (Pye Unicam SP6-500) (or translated f r o m cell numbers if a standard curve was available). One ml of subsample was withdrawn from each of the culture flasks usually on the 2rid, 6th and 10th day. Cell numbers were counted in a Sedgwick-Rafter counting chamber. Between the 13th and 14th day, when Chlorella biomass reached the m a x i m u m yield plateau, the algal cultures were terminated and final optical density readings were obtained. After growth curves were constructed, the specific growth rate was computed by the equation ln(x2/xl)/(t2-t !) where Xl and x2 are cell concentrations counted on day 6(t2) and day 2(tl).
Assays o f Triton X-IO0 To examine the effect of Triton on Chlorella cells in assays, a stock solution of Triton X-100 was prepared by dissolving 1.0 ml of the surfactant in 50 ml of deionized distilled water. Using 10°70 Bristol's solution as the growth medium, Triton concentrations were made up to 0.2, 0.4 and 0.8 m M strength in the test solutions, with zero percent Triton as a control. Chlorella cells were inoculated and growth response was recorded.
Assays o f Triton X-IO0 with metals To examine the effect of Triton with metals on Chloreila cells, stock solutions of
118
CuC12" 2 H 2 0 and Cd(NO3)2" 4 H 2 0 were made up to a concentration of 0.5 g ' l A screening test for both metals was conducted at metal concentrations of 0.05, 0.5 and 5.0 mg" 1- ~ for 5 days. According to the test results, final metal concentrations (0.05, 0.25, 0.5, 1.0 and 1.25 m g . l - 1 for C d 2 + , for example) were prepared with 10°70 Bristol's solution and to each of these concentrations 0.4 m M Triton was added. (0.4 mM had been observed to have sufficient strength to prevent coagulation of Chlorella cells in most natural waters in the preliminary tests.) Samples containing identical metal concentrations without Triton added were used as controls. Chlorella cells were inoculated and assay procedures were followed as before.
Assays of Triton X-IO0 with organic compounds To study the effect of Triton with organic compounds on Chlorella growth, hyamine 3500* (an algicide), pentachlorophenol (PCP), p-chlorophenol, formaldehyde and the herbicide (Crabgrass Killer FMC, a 2,4-D complex) were used in the assays. The water-insoluble p-chlorophenol and P C P were dissolved in 5 ml of acetone, which were then made up to a liter stock solution of 100 g g - l - ~ while the other compounds were made up to 1.0 mg.1-1 or 1:100 dilution with distilled water. Acetone was kept below 0.05% in the final test solutions to avoid causing injuries to Chlorella cells (Schanberger and Wildman, 1977; Wong et al., 1982). Prior to the test, the toxicity range for each of the test compounds was screened using widely spread concentrations as described for that of metal testing. Definitive concentrations were then chosen. Experiments were carried out in 10% Bristol's medium with or without the addition of 0.4 m M Triton. The algal inoculation procedure was as before. The Triton-organics pairing effect on Chlorella cells was further investigated with hyamine at different Triton concentrations. To each hyamine dosage of 0.32, 0.80, 1.6 and 2.4 m g - 1 - ~ in 10% Bristol's, a sequence of Triton concentrations, i.e., 0.1, 0.2, 0.4, 0.8 and 1.2 mM, was added. Assay procedures were carried out as before. Chlorella biomass was measured by reading the optical density of the culture medium at the end of the incubation period. Results are presented as the mean. Relationships between treated and control samples were investigated using linear regression analysis while their differences were evaluated using the t-test for match pairs and the significance values reported at the P < 0 . 0 5 level. RESULTS
Routine assays with natural waters The water qualities of the study lakes are described in Table I. The remote lakes *Hyamine 3500, an 80°70 concentrate in ethanol, has an active ingredient n-alkyl (50% CI4, 40% C12, 10070 C~6) dimethylbenzylammoniumchloride.
Heart L.
Penetang Bay
Gravenhurst Bay
Urban lakes Aquitaine Reservoir
Trout L.
Swan L.
Ruth Roy L.
Plastic L.
Miskokway L.
McCann L.
Heeney L.
Chub L.
Remote lakes Bowland L.
Study lakewaters
43°35 ' N 79045 ' W 44°55'N 79°24'W 44°46'N 79o56'W 43°44'N 79047 ' W
47o05 ' N 80 °50' W 45 ° 13' N 78o59 ' W 45°08'N 79 ° 06' W 45°45'N 79 ° 10' W 45°39'N 80o14'W 45 ° 11' N 78o50'W 46°05'N 81o14'W 46o22 ' N 81o04'W 45°35 ' N 80 °04' W
Coordinates (Lat. (°N) Long. (°W))
relaorted by Suns et al. (1980).
7.6
8.0
7.1
8.1
6.2
5.1
4.8
6.2
6.0
6.3
5.9
5.5
5.4
pH
102.0
85.0
17.0
146.0
15.0
15.0
6.0
7.0
10.0
N/A
7.0
8.0
16.0
Hardness (CaCO3 mg • 1- l)
0.087
0.023
0.014
0.046
0.004
0.005
0.001
0"005
0.005
N/A
0.005
0.005
0.003
Total phosphorus (TP) (mg • 1- i)
1.11
0.33
0.34
0.65
0.20
0.23
0.12
0.31
0.35
N/A
0.24
0.28
0.20
Total Kjeldahl nitrogen (TKN) (mg • 1- i)
9.6
3.3
4.2
3.6
3.9
1.1
0.5
2.4
3.6
N/A
2.5
4.4
2.1
Dissolved organic carbon (DOC) (mg • 1- l)
Gravenhurst Bay PCB 350+ 85 ~ D D T 83 + 14 Dieldrin 5 + 2 Chlordane 3+_1 DDE
Chub L. PCB trace DDE trace
Heeney L. PCB 256 + 84 DDE 20
Organic residues in yearling yellow perch (ng• g - i wet wt.)
Some water quality characteristics of the 13 study lakes. The mean concentrations of organic residues analysed from the fatty tissues of the yearling perch
TABLE I
120
situated in the precambian Shield, about 200 km north of Toronto, are generally unproductive, with total phosphorus concentrations below 0.005 mg-1- 1 and total Kjeldahl nitrogen concentrations below 0.35 mg-1-1. These waters are low in CaCO3, between 6 and 22 m g - l - 1 and their pH often fails below 6.3. The urban lakes, on the contrary, are enriched waters with high nutrients (TP between 0.046 and 0.087 mg-I - 1 and TKN between 0.33 and 1.11 mg.l-1), high CaCO3 concentrations (between 85 and 146 mg. 1-1) and high pH values (7.6-8.1) except Graverhurst Bay which is more of a softwater urban lake with CaCO3 hardness of 17 mg.1The Chlorella growth curves observed from routine assays with these enriched, filtered lakewaters portray two kinds of algal response (Fig. 1). The first kind represented by Chub L. (a humic lake) and Stream A (entering Plastic L.) shows a normal pattern of Chlorella growth; the algal biomass increases during the early days of incubation to attain a logarithmic growth phase, then the growth slope gradually levels off to establish a maximum yield plateau when a certain limiting factor sets in. The second kind, denoted by the annihilation of Chlorella growth in the cultures as depicted by Miskokway L. and Heart L. in the figure, occurs rather frequently in our study in both the hard and soft waters. During incubation, Chlorella cells in these water media were quite insensitive to the addition of the chelator, NazEDTA; they formed clusters sometimes of up to a hundred ceils or more.
10 7 Chub Lake (humic water) Stream A (Plastic Lake) o Heart Lake [hardwater) • Miskokway Lake (softwater) []
106
T
E 03 J ,...J LL]
0 < J
k~ 105 122
0
I
0
104
I
I
I
I
I
I
I
2
4
6
8
10
12
14
DAYS
Fig. 1. Growth response of Chlorella cells in natural water media enriched with 10% Bristol's solution.
121
Organic contaminants were undetected in the water analysis. The detection limits for certain compounds such as PCB, DDT and chlordane for example are 20, 5 and 2 ng.l-a, respectively. The dissolved organic carbon concentrations (DOC) were also low, ranging between 0.5 and 4.4 mg.1-1 except for Heart L. (Table I). Although the organic trace contaminants had concentrations far below the detection limits from the water analysis, Suns et al. (1980) from their study of the bioaccumulation of organics in fish tissues, illustrated substantial quantities of PCB, DDT, chlordane and dieldrin embedded in the fatty tissues of yearling yellow perch from three of our study lakes (Table I). The PCB concentrations of between 256 and 350 ng.g-1 were the most prominent among the tested compounds in these waters. Response of Chlorella cells to Triton X-lO0 The growth of Chlorella cells did not show significant changes with the addition of Triton X-100 in 10°70 Bristol's medium (Fig. 2). The specific growth rates calculated were not significantly different among the treated samples and the con107
o
control
• 4- 0.2mM Triton X-100 v "1- 0.4raM Triton X-100 D-I-0.8mM Triton X-t00
E
106
o z
o I-< rr I-IZo
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sp. gr. rate (d"1)
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T=I= S.E. 104
control
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2.0 x 1064"0.1 x 106
+0.2raM Triton X-100
0.54"+'0.05
2.3 x 106--+0.5 x 106
+0.4mM Triton X-100
0.53"1-0.13
1.5 x 1064"0.2 x 106~
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1.6 x 1064"0.3 x 106~
I
t
I
t
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t
2
4
6
8
10
12
14
DAYS Fig. 2. G r o w t h response o f Chlorella ceils in 10% Bristol's m e d i u m with and without Triton X-100 addition. *Indicates significant differences ( P < 0 . 0 5 ) f r o m the control; n = 3.
122
trol (inset, Fig. 2). Very slight drops in Chlorella yields were observed in Triton treated samples o f 0.4 and 0.8 raM. Although the decrease in biomass appeared to be negligible, there was a significant difference ( P < 0.05) between the sample means o f the control and the Triton treated cultures.
Pairing effect of Triton X-IO0 and metals on Chlorella cells Chemically, Triton produced a synergistic effect o f metal toxicity on Chlorella cells. When mixed with 0.4 mM Triton at low Cu 2+ concentrations, growth of Chlorella cells appeared to be stimulated; but at higher Cu 2 ÷ concentrations o f around 0.04 mg" 1- 1, Cu 2 + toxicity was enhanced by Triton with complete inhibition of Chlorella growth observed at about 0.065 mg Cu z + .1-1 (Fig. 3a). The synergistic effect was more obvious when Triton was paired with Cd 2+. Chlorella growth decreased appreciably in the Cd 2 + cultures when Triton was added (Fig. 3b). Since Cd 2÷ is not as detrimental as Cu 2+ (Wong and Beaver, 1981), it
0.5 -
J
a)
• Cu
.+
-~-0.4mM Triton X-IO0
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0.3 0.2
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0.50
0.75
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Cd** [mg I- t ] Fig. 3. G r o w t h response o f Chlorella cells to (a) Cu 2 + and (b) Cd z ÷ with and without the presence o f T r i t o n X-100 in 10% Bristol's medium.
123 v p-chlorophenol-4- 0.4mM Triton X- 100 0.6 t
0.4
a)
=p-chtorophenol
~...~~.~ •
?
, v ~
0.2
0
I
I
I
I
5
10
15
20
p-CHLOROPHENOL (mg r t) 0.6 t
•
b)
pentachloroPhenol-1-O.4mM Triton X-lO0
opentachloroghenol 0.4 'O) >, ¢0 LU I-<
Q:
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P E N T A C H L O R O P H E N O L ( m g I-t) 0.6
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aHerbicide-t-0.4mM
LL
0.4
0.2
.0125
.0250
HERBICIDE (CRABGRASS 0.6
d)
.0375
.0500
KILLER %)
• Formaldehyde 4" 0.4mM Triton X- 100 OFormaldehyde
0,4
0.2
~;
0
.002
.004
I
.006
n
I
.008
FORMALDEHYDE (%) Fig. 4. Growth response of Chlorella cells to (a) p-chlorophenol, (b) pentachlorophenol, (c) herbicide (Crabgrass Killer FMC), and (d) formaldehyde with and without the addition of Triton X-100 in 10% Bristol's medium.
124
took 1.25 mg" 1- a of Cd z + (about 20 times the inhibit growth of Chlorella cells.
Cu 2 +
concentration) to completely
Pairing effect o f Triton X-IO0 and organics on Chlorella cells In contrast to its synergistic effect with metals, Triton when combined with organic compounds was found to have an ameliorating effect on Chlorella growth (Fig. 4). At 0.4 mM, Triton was capable of relieving part of the toxic stress on Chlorella cultures exposed to formaldehyde and the herbicide (Crabgrass Killer) (Fig. 4c, d). When coupled with pentachlorophenol and p-chlorophenol, Triton showed a more complete antagonistic reaction with these compounds thus allowing better growth of Chlorella cells in the assays (Fig. 4a, b). Triton's growth revitalizing ability with organics was most pronounced when paired with hyamine, an algicide. By using maximum algal yield as the measuring parameter, it was observed from a sequence of Triton concentrations that a better Chlorella yield ensued with every increase in concentration of Triton in the medium until the biomass finally reached its maximum yield plateau (Fig. 5a). Then from the biomass yield curves constructed in Fig. 5a, the binding concentration of Triton or the amount of Triton required to immobilize the hyamine activity 25 o 0.32 rng hyamine 1-1 a)
b)
• 0.80 rng hyamine i -1 1.60 mg hyamine 1-1
20
= 2.40 mg hyarrline I"1
,.._..
E
o
10
0
% binding
£3
d LLI >-
10
/
klA
z
conc~t;tion
.< >.. -r-
< d ..d LLI cc
1.0
0 -i0
,.._1
0 0
0.5 TRITON
X-tO0
,
!
1.0
1.5
(mM)
0.1
I
I
I
0.5
1.0
1.5
TRITON X- 100 (raM)
Fig. 5. (a) Chlorella biomass curves constructed from yields obtained from various combinations of hyamine and Triton X-100 pairings. The ascending slope was extrapolated to intercept at the extended maximum yield plateau for the estimation of the binding concentration of Triton or the amount of Triton required to immobilize all organic activities in the medium. (b) The relationship between the binding concentration of Triton and the hyamine dosage used.
125
in the medium can be estimated by extrapolating the ascending slope of the biomass curve to intercept the line extended from the maximum yield plateau as demonstrated in the figure. Further, by plotting the binding concentration of Triton against the hyamine dosage used, a relationship between Triton and hyamine can be established to estimate the toxic stress of a test water sample in terms of hyamine dosage if the amount of Triton required to achieve a Chlorella maximum yield plateau is known (Fig. 5b). Evaluation of toxicity o f trace contaminants in waters Finally, when assays were tested with enriched natural waters treated with Triton, Chlorella cells that were previously growth inhibited (such as those in control cultures with zero Triton in Figs. 6 and 7) became activated and recovered steadily
REMOTE LAKES ~ B o w l a n d Lake 0.4
v C h e b Lake i Heeney Lake "
•
uMoOann
Iake
eMiskokway I
Lake
oPlastic Lake ~Ruth Roy Lake
binaing 0.3
~Swan Lake oTrout Lake
concentration
0.2
¢D
E
0
o.1
0 0
i
I
I
I
I
I
I
0.2
0.4
0.6
0.8
t .0
1.2
1.4
T R I T O N X-100 (mM) Fig. 6. Chlorella b i o m a s s versus Triton X - I 0 0 for remote lakes. Z e r o Triton concentration was used as a control.
126 0.3 URBAN LAKES ~' Aquitaine Reservoir • GravenhurstBay o Heart Lake 0"3 .<
a PenetangBay
0.2
O
Z W a d 0.1 <
binding concentration
~
~
I--13_ O
0 0
0.2
0.4
0.6
0.8
1.0
1.2
I 1.4
TRITON X-100 [mM] Fig. 7. Chlorella biomass versus Triton X-100 for urban lakes. Zero Triton concentration was used as a control.
when accompanied by increasing Triton concentrations until the maximum yield plateau was attained. When the biomass curves o f the test waters were compared, T A B L E II The trace organic toxic stresses translated into mg h y a m i n e . l - ~ for our study lakes. Study lakewaters
Organic toxic stress (mg hyamine • 1-)
Estimated binding conc. of Triton (mM)
Remote lakes Bowland L. Chub L. Heeney L. M cCan n L. M i s k o k w a y L. Plastic L. Ruth Roy L. Swan L. Trout L.
0.7 0.0 0.8 0.8 0.8 0.5 2.3 1.5 0.8
0.40 0.00 0.50 0.45 0.48 0.24 1.00 0.80 0.46
Urban lakes Aquitaine Reservoir Gravenhurst Bay Penetang Bay Heart L.
1.0 1.5 1.0 0.7
0.62 0.80 0.60 0.40
127 the urban lakes showed markedly lower biomass plateaus than those of the remote lakes. Following the approach described earlier, the binding concentrations of Triton which permit maximum Chlorella yields were estimated. The binding concentration for Miskokway L., for example, as constructed by the dotted lines (Fig. 6) was estimated to be 0.5 mM Triton and according to the Triton-hyamine curve in Fig. 5b, the organic toxic stress was translated as 3.6 mg hyamine.lThe toxicity of trace organic contaminants for our study lakes is translated in Table II. The highest stress was obtained from Ruth Roy L. at about 2.3 mg hyamine.1-1 and the lowest was from Plastic L. at about 0.5 mg hyamine-1-1 These estimated organic toxic stresses, however, showed no correlation with the DOC measurements of the lakewaters. DISCUSSION Organic contaminants have been suspected in many lakes in southern Ontario when Chlorella assay cultures failed repeatedly with treatments of both nutrients and the chelator Na2EDTA. Somehow, because of extremely low levels, mostly in the ng' l- i range, contaminants such as PCB and DDT have not been perceived as significant growth inhibitors of biotic communities thus far. Only in more recent studies have scientists begun to realize through hioaccumulation studies of living tissues such as fish and clams, and from the analysis of sediments in urban and remote lakes, that many trace organic compounds such as polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB) and insecticides are, in fact, prevalent in aquatic environments and are a potential threat to many sensitive species (Hamelink et al., 1975; Addison, 1976; Butler and Schutzmann, 1977; Tan and Heit, 1981; Tsui and McCart, 1981; Gooch and Hamdy, 1983; Niimi, 1983). Contaminants in water are absorbed by fish and accumulated in their tissues. Suns et al. (1980) demonstrated magnified concentrations of PCB, DDT, chlordane and dieldrin residues in yearling perch from three of our study lakes. Perhaps the bioavailability of these trace organic substances helps to explain why growth of Chlorella cells was inhibited in assays with these waters and also why Chlorella cells formed clusters instead of forming enlarged, single deformed cells similar to those observed in tests with metals. Many surfactants, especially those in the octylphenol and nonylphenyl series, are effective binding substances for organic compounds in solutions besides serving as emulsifiers or surface active agents. At high concentrations, Triton can effectively retain organic residues by aggregating the hydrophobic portion of the molecules in groups and keep them inactive in the internal region of the micelles (Swisher, 1970; Abel, 1974). There are definitely a number of other organic polymers that have a great affinity for organic compounds (Gustafson and Paleos, 1971; Healy, 1971;
128 Dressier, 1979) but among our tested nonionic surfactant series of polyoxyethyleneglycol (Tween) and polyethoxyethanol (Triton), Triton X-100 appears to be the only effective agent which by itself exerts little harmful effect on Chlorella cells in the assays. At low concentrations, as reported in the literature, Triton has not been found to hamper the growth process of living cells except those of bacteria and virus cells mentioned by Simmons et al. (1977). Triton has no effect on the glucose oxidative process of human lymphocytes (Hrabak, 1982); it does not impede germination of Petunia pollen grains (Pfahler et al., 1981); it only slightly moderates Chlorella growth in our study. Above all, it is important that Triton does not serve as a growth stimulant (Fig. 2) or as a source of organic carbon to Chlorella cells. By solubilizing the protein and lipid layers in cell membranes as suggested by Capaldi (1977) and Ukekes (1965), Triton probably enhances large influxes of metal into the cells and thus magnifies their toxicity in the cultures. But chemically, Triton does not react with metals such as Cu 2 + in solution. During the polarographic calibration of Cu 2÷ when 0.01% of Triton was immersed in the metal solution, there was no significant change observed in the calibrated curve of Cu 2 + nor was there any apparent reduction in the height of the maximum, except the smoothing out of the curve which is generally regarded as a physical phenomenon. On the other hand, because of the micellar interaction, Triton restricts the mobility of organic compounds such as PCP, p-chlorophenol, formaldehyde and hyamine, and prevents them from interacting with the Chlorella cells such that when sufficient Triton is available in the medium, Chlorella cells, protected from the organic toxicants, are capable of attaining their maximum yield at the growth plateau (Fig. 5a). Chlorella cells responded favourably to enriched natural water media once Triton was introduced (Figs. 6 and 7). On the assumption that organic residues were present in trace quantities in these waters as evidenced by Suns et al. (1980) and also Triton could immobilize activities of organic materials, the recovery of Chlorella cultures in our assays suggests the predominance of organic toxicity in these waters. If metals were the controlling factor, the treatment of these waters with Triton would have only deepened the growth depression and certainly not revived the growth of Chlorella cells. Once the organic stresses in the water systems were quantitatively estimated, as shown in Table II, a better interpretation of the water quality of these waters could be made even just by comparing these data alone; for example, urban lakewaters suggested a higher burden of organic wastes while the remote lakes, on the whole, were less affected by organic pollutants except Swan L. and Ruth Roy L. which showed high toxicity values. Containing abundant suspended matter, Chub, the humic lake, was observed to be free from organic toxic stresses. It is obvious that there are other growth inhibitors, e.g., metal complexes, pH, etc., present in these waters, particularly those of the urban lakes which showed ex-
129
tremely low yields in the biomass plateaus (Fig. 7). Only by integrating this method with the approaches we suggested earlier on total toxicity and metal mixtures, may we perhaps develop a better understanding of the inorganic fraction of toxicity in these waters (Wong and Beaver, 1980; Wong, 1984). In conclusion, the bioaccumulation of organic residues in fish tissues suggests the bioavailability of trace contaminants that are not easily detectable in routine water analysis. Triton X-100, effectively reducing the toxic effect of single test compounds in Chlorella assays, presumably behave similarly to immobilize molecules of organic mixtures in natural waters. Despite its positive ameliorating growth effect with organic compounds on Chlorella, Triton could only mitigate a toxic effect no greater than an equivalent of about 2.4 mg hyamine • 1- 1 in the cultures. Biomonitoring, whether with fish or clams, is a good device to detect the presence of trace toxicants but it will be more valuable if the toxicity of the organic trace mixtures can be quantified at the same time. It is hoped that the bioassay approach suggested above will have some use in future assessment of organic toxicants in natural waters. REFERENCES Abel, P.D., 1974. Toxicity of synthetic detergents to fish and aquatic invertebrates. J. Fish. Biol. 6, 279-298. Addison, R.F., 1976. Organochlorine compounds in aquatic organisms: their distribution, transport and physiological significance. In: Effects of pollutants on aquatic organisms, edited by A.P.M. Lockwood, Cambridge Univ. Press, London, pp. 127-144. Alfheim, I., J. Gether and G. Lunde, 1978. Access of some atmospheric non-polar organic micropollutants to lakes and rivers. Acid precipitation - effects on forest and fish project. Res. Report 14, Norway. Anon., 1981. Triton surface-active agents. Rohm and Haas, Philadelphia, PA, 28 pp. American Public Health Association, 1980. American Water Works Association and Water Pollution Control Federation, Bioassay methods for aquatic organisms. In: Standard method for the examination of water and waste-water, 15th edition, edited by A.E. Greenberg, J.J. Connors and D. Jenkins, APHA, Washington, DC, 615 pp. Blaise, C., R. Legault and N. Bermingham, 1983. A simple microassay technique for measuring algal growth inhibition (ECs0's) in aquatic toxicity studies. Proceeding of the 9th annual aquatic toxicity workshop, Nov. 1-5, 1982. Edmonton, Alberta, edited by W.C. McKay, Canadian Technical Report of Fish. and Aquat. Sci. No. 1163, pp. 1-8. Butler, P.A. and R.L. Schutzmann, 1977. Bioaccumulation of DDT and PCB in tissue of marine fish. ln: Aquatic toxicology, edited by L.L. Marking and R.A. Kimerle, ASTM 667, Philadelphia, PA, pp. 212-220. Capaldi, R.A., 1977. The structural properties of membrane proteins. In: Membrane protein and their interactions with lipids, edited by R.A. Capaldi, Plenum, NY, pp. 1-20. Cooke, M. and A.J. Dennis, 1981. Chemical analysis and biological fate: polynuclear aromatic hydrocarbon, 5th Int. Syrup., Battelle Press, 770 pp. Cruickshank, J.H., 1968. Industrial uses of non-ionic surface-active agents. Water Pollut. Control. Symposium paper 5, pp. 100-107. Donaldson, W.T., 1977. Trace organics in water. Env. Sci. Technol. 11, 348-351. Dressier, M., 1979. Extraction of trace amounts of organic compounds from water with porous organic polymers. J. Chromatography 165, 167-206.
130 Ferrard, J.F., P. Vasseur and J.M. Jouany, 1983. Value of dynamic tests in acute ecotoxicity assessment in algae. Proceedings of the 9th annual aquatic toxicity workshop, Nov. 1-5, 1982. Edmonton, Alberta, edited by W.C. MacKay, Canadian Technical Report of Fish. and Aquat. Sci. No. 1163, pp. 38-56. Geyer, H., G. Politzki and D. Freitag, 1984. Prediction of ecotoxicological behaviour of chemicals: relationship between n-octanol/water partition coefficient and bioaccumulation of organic chemicals by alga Chlorella. Chemosphere 13, 269-284. Gooch, J.A. and M.K. Hamdy, 1983. Uptake and concentration factor of Aroclor 1254 in aquatic organisms. Bull. Environ. Contam. Toxicol. 31, 445-452. Gustafson, R.L. and J. Paleos, 1971. Interactions responsible for the selective adsorption of organics on organic surfaces. In: Organic compounds in aquatic environments, edited by S.J. Faust and J.V. Hunter, Marcel Dekker, Inc., NY, pp. 213-238. Hamelink, J.E., R.C. Waybrant and P.R. Yant, 1975. Mechanism of bioaccumulation of mercury and chlorinated hydrocarbon pesticides by fish in lentic ecosystems. In: Fate of pollutants in the air and water environments, edited by 1.H. Suffer, Wiley-lntersci., pp. 261-281. Healy, T.W., 1971. Selective adsorption of organics on inorganic surfaces. In: Organic compounds in aquatic environments, edited by S.J. Faust and J.V. Hunter, Marcel Dekker, Inc., NY, pp. 187-212. Hrabak, A., 1982. Damaging effect of detergents on human lymphocytes. Bull. Environ. Contain. Toxicol. 28, 504-511. Hutchinson, T.C. and P.M. Stokes, 1975. Heavy metal toxicity and algal bioassays. Amer. Soc. for testing and materials. Special Tech. Publ. 573, pp. 321-343. International Organization for Standardization (ISO), 1980. Algal growth inhibition test, draft method, Delft, The Netherlands. Joubert, G., 1980. A bioassay application for quantitative toxicity measurement using the green algae Selenastrum capricornutum. Water Res. 14, 1759-1763. Lillie, T.H., R.E. Hampson, Y.A. Nishioka and M.A. Hamilton, 1982. Effectiveness of detergent and detergent plus bleach for decontaminating pesticide applicator clothing. Bull. Environ. Contain. Toxicol. 29, 89-94. Lunde, G. and A. Bjorseth, 1977. Polycyclic aromatic hydrocarbons in long-range transported aerosols. Nature 268, 518-519. Metcalf, R.C., 1977. Biological fate and transformation of pollutants in water. In: Fate of pollutants in the air and water environments, edited by i.H. Suffet, Wiley-lnterscience, pp. 195-221. Niimi, A.J., 1983. Biological and toxicological effects of environmental contaminants in fish and their eggs. Can. J. Fish. Aquat. Sci. 40, 306-312. Organization for Economic Cooperation and Development (OECD), 1981. Guidelines for testing of chemicals, Paris, France, 201 pp. Payne, A.G. and R.H. Hall, 1979. A method for measuring algal toxicity and its application to the safety assessment of new chemicals. In: Aquatic toxicology, edited by L.L. Marking and R.A. Kimerle, ASTM 667, Philadelphia, PA, pp. 171-180. Pfahler, P.L., H.F. Linskens, H.W. Schoot and M. Wilcox, 1981. Surfactant effects on Petunia pollen germination in vitro. Bull. Environ. Contam. Toxicol. 26, 567-570. Schauberger, C.W. and R.B. Wildman, 1977. Accumulation of aldrin and dieldrin by blue-green algae and related effects on photosynthetic pigments. Bull. Environ. Contam. Toxicol. 17, 483-488. Simmons, K., H. Garoff and A. Helenius, 1977. The glycoproteins of the semliki forest virus membrane. In: Membrane proteins and their interactions with lipids, edited by R.A. Capaldi, Plenum, NY, pp. 207-234. Starr, R.C., 1,964~ The culture collection of algae at Indiana University. Amer. J. Bot. 51, 1013-1044. Suns, K., C. Curry and D. Wilkins, 1980. The effects of road oiling on PCB accumulation in aquatic life. Ontario Min. of Environ., 14 pp. Swisher, R.D., 1970. Surfactant biodegradation. Marcel Dekker, Inc., NY, 496 pp. Tan, Y.L. and M. Heit, 1981. Analysis of polynuclear aromatic hydrocarbons in sediment cores from
131 two remote Adirondack lakes by resolution gas chromatography/mass spectrometry. In: Polynuclear aromatic hydrocarbons. 5th Int. Symp., Battelle Press, pp. 561-570. Tsui, P.T.P. and P.J. McCart, 1981. Chlorinated hydrocarbon residues and heavy metals in several fish species from the Cold Lake area in Alberta, Canada. Inter. J. Env. Anal. Chem. 10, 277-285. Ukeles, R., 1965. Inhibition of unicellular algae by synthetic surface active agents. J. Pt~ycol. 1,102-110. U.S. Environmental Protection Agency, 1978. The Selenastrum capricornutum Printz. algal assay bottle test: experimental design, application and data interpretation protocol. USEPA, Environmental Research Laboratory, Corvallis, OR, 126 pp. Wong, S.L., 1984. Toxicity and water quality: a bioassay interpretation. J. Env. Sci. Health 19, 377-386. Wong, S.L. and J.L. Beaver, 1980. Algal bioassays to determine toxicity of metal mixtures. Hydrobiol. 74, 199-208. Wong, S.L. and J.L. Beaver, 1981. Metal interactions in algal toxicology: conventional versus in vivo tests. Hydrobiol. 85, 67-'/1. Wong, P.T.S., Y.K. Chau, O. Kramar and G.A. Bengert, 1982. Structure-toxicity relationship of tin compounds on algae. Can. J. Fish. Aquat. Sci. 39, 483-488. Zoetman, B.C.J., K. Harmsen, J.B.H.J. Linders, C.R.H. Morra and W. Slooff, 1980. Persistant organic pollutants in river water and ground water of the Netherlands. Chemosphere, 9, 231-249.
115
AQT 00146 ALGAL
ASSAY EVALUATION
FACE WATER
OF TRACE
USING THE NONIONIC
CONTAMINANTS
SURFACTANT,
IN SUR-
TRITON
X-100
S.L. WONG
Ontario Ministry o f the Environment, Aquatic Contaminants Section, Water Resources Branch, P.O. Box 213, Rexdale, Ontario M g w 5LI, Canada (Received 2 April 1984; revised version received and accepted 4 December 1984)
Triton X-100, a nonionic surfactant, has been found to be capable of neutralizing the toxic effect of selected organic compounds in algal assays using 10o7o Bristol's medium. This nonionic surfactant does not seem to affect specific growth rates of Chlorella cultures when it is added alone to the medium but it accelerates growth inhibition when coupled with metals. By administering a series of increasing Triton concentrations in assays with natural waters, Chlorella growth that was once arrested in the test waters became revitalized and growth was more profuse as the concentration of Triton increased. The results indicate that Triton has the ability to alleviate the toxic stresses of organic mixtures that are contaminating many southern Ontario lakes. The bioavailability of these trace organics such as PCB, DDT, chlordane and dieldrin was confirmed in the study lakewaters from bioaccumulation in fish tissues. Key words: trace organic contaminants; metals; natural waters; algal assay; surfactant; Chlorella fusca
INTRODUCTION
Many lakewaters in southern Ontario were found to be incapable of supporting algal growth in assays when they were used as culture media (Wong, 1984). After repeated attempts failed to initiate Chlorella growth with both nutrients and the chelator, NazEDTA, it appeared that the inhibition of algal growth was much more than just the deficiency of nutrients or the toxicity of metals. Organic trace contaminants were therefore suspected. The following study was undertaken to test the hypothesis that the inhibition of algal growth in these assays might have been due to stress from organic contaminants prevalent in aquatic environments (Donaldson, 1977; Lunde and Bjorbeth, 1977; Metcalf, 1977; Alfhiem et al., 1978; Zoetman et al., 1980; Cooke and Dennis, 1981). So far, there has not been an appropriate method developed for assessing trace organic contaminants in natural waters despite the many algal toxicity assay techniques available - some are well developed (USEPA, 1978; APHA, 1980; ISO, 1980; OECD, 1981) and some have greatly improved (Payne and Hall, 1978; Joubert, 1980; Blaise et al., 1983; Ferrard et al., 1983). 0166-445X/85/$03.30
© 1985 Elsevier Science Publishers B.V. (Biomedical Division)
116
Apparently, toxicity assay methods developed thus far, are mostly concerned with the management of industrial wastewaters. Since it is essential to know the amount of dilution needed to lower the toxicity of a waste effluent to an acceptable level, the dilution technique, to produce an LCs0 value (lethal concentration), becomes the backbone of conventional approaches. However, surface waters on the other hand, contain numerous trace contaminants, with some 500-700 new organic chemicals entering each year (Metcalf, 1977). The contaminants, though trace in amount, can be highly toxic at times, e.g., dioxin. It is therefore difficult to determine, by employing the dilution technique, how m a n y toxic substances are being 'washed away' or by how much water quality has been changed. Most of all, dilution is not a technique used to single out organic toxicity in water. Therefore, in order to evaluate the organic toxic stress in these waters and to quantify the growth inhibition of algae in the system, an alternative algal toxicity test has been suggested in this paper for the examination of trace contaminants. MATERIALS AND METHODS
Test organism The green, unicellular alga, Chlorellafusca Shihers et Krauses obtained from the C a n a d a Centre for Inland Waters in Burlington, Ontario, was used as the test organism in this study. C. fusca showed high tolerance to metals (Wong and Beaver, 1980), organics (Geyer et al., 1984) and low p H (Hutchinson and Stokes, 1975) in assays. Unlike the universal test alga, Selenastrum capricornutum which is very sensitive to toxicant, Chlorella often achieves sufficient biomass for tissue analysis of contaminants. It is preferred as a test alga in a toxicity study such as this. Chlorella cultures were maintained in 10% Bristol's solution (Starr, 1964) and were used as an inoculum only when the cells reached the logarithmic phase of growth, that is between the 7th and 10th day.
Triton )(-100, a specific test compound Triton X-100 (isooctylphenoxypolyethoxyethanol), a nonionic surface active agent, has the following structural formula:
R"~ ~"O(OCH2CH2)10OH It is biodegradable and water soluble and it contains an average of 10 mol ethylene oxide in the octylphenol series. It emulsifies and solubilizes organic solvent in solution and has been used to remove lipid, hydrocarbon and other organic stains by textile industries (Cruickshank, 1968; Anon., 1981; Lillie et al., 1982).
117
Assays o f natural waters Surface water samples were collected f r o m 13 lakes, reservoirs and bays in southern Ontario in the summer of 1981 and 1982 (Table I). Each sample was separated into two parts on arrival at the laboratory. One part was for routine chemical analyses which included p H , hardness, dissolved organic carbon (DOC), total phosphorus (TP) and total Kjeldahl nitrogen (TKN); the other part was used for algal toxicity tests. Water samples were filtered through a 0.45 #m membrane filter and the Erlenmeyer flasks (250 ml) were acid washed before use. The assay was carried out by dividing a filtered water sample into seven 90-ml portions with duplicates. Ten ml of normal Bristol's solution were added to each portion to provide a 10°70 Bristol's medium so as to eliminate any possible deficiency o f nutrients, especially those from the softwater lakes (hardness<20 mg CaCO3" 1- 1). The first portion was used as a control; the second portion, to which 10 m g . l - 1 Na2EDTA were added, was used for evaluation o f metal toxicity; the 3rd, 4th, 5th, 6th and 7th portions contained Triton X-100 in concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0 m M , respectively. One ml of Chlorella cells was inoculated into each flask to give an initial concentration o f about 8 × 104 cells per ml. The culture flasks were mounted on a platform shaker set at 90 oscillations per min. The cultures were illuminated on a 12:12 light-dark cycle by cool, white fluorescent light at about 8000 lux and were kept at temperatures between 18-20°C. The optical densities of the initial cell concentrations were read at 730 nm with a spectrophotometer (Pye Unicam SP6-500) (or translated f r o m cell numbers if a standard curve was available). One ml of subsample was withdrawn from each of the culture flasks usually on the 2rid, 6th and 10th day. Cell numbers were counted in a Sedgwick-Rafter counting chamber. Between the 13th and 14th day, when Chlorella biomass reached the m a x i m u m yield plateau, the algal cultures were terminated and final optical density readings were obtained. After growth curves were constructed, the specific growth rate was computed by the equation ln(x2/xl)/(t2-t !) where Xl and x2 are cell concentrations counted on day 6(t2) and day 2(tl).
Assays o f Triton X-IO0 To examine the effect of Triton on Chlorella cells in assays, a stock solution of Triton X-100 was prepared by dissolving 1.0 ml of the surfactant in 50 ml of deionized distilled water. Using 10°70 Bristol's solution as the growth medium, Triton concentrations were made up to 0.2, 0.4 and 0.8 m M strength in the test solutions, with zero percent Triton as a control. Chlorella cells were inoculated and growth response was recorded.
Assays o f Triton X-IO0 with metals To examine the effect of Triton with metals on Chloreila cells, stock solutions of
118
CuC12" 2 H 2 0 and Cd(NO3)2" 4 H 2 0 were made up to a concentration of 0.5 g ' l A screening test for both metals was conducted at metal concentrations of 0.05, 0.5 and 5.0 mg" 1- ~ for 5 days. According to the test results, final metal concentrations (0.05, 0.25, 0.5, 1.0 and 1.25 m g . l - 1 for C d 2 + , for example) were prepared with 10°70 Bristol's solution and to each of these concentrations 0.4 m M Triton was added. (0.4 mM had been observed to have sufficient strength to prevent coagulation of Chlorella cells in most natural waters in the preliminary tests.) Samples containing identical metal concentrations without Triton added were used as controls. Chlorella cells were inoculated and assay procedures were followed as before.
Assays of Triton X-IO0 with organic compounds To study the effect of Triton with organic compounds on Chlorella growth, hyamine 3500* (an algicide), pentachlorophenol (PCP), p-chlorophenol, formaldehyde and the herbicide (Crabgrass Killer FMC, a 2,4-D complex) were used in the assays. The water-insoluble p-chlorophenol and P C P were dissolved in 5 ml of acetone, which were then made up to a liter stock solution of 100 g g - l - ~ while the other compounds were made up to 1.0 mg.1-1 or 1:100 dilution with distilled water. Acetone was kept below 0.05% in the final test solutions to avoid causing injuries to Chlorella cells (Schanberger and Wildman, 1977; Wong et al., 1982). Prior to the test, the toxicity range for each of the test compounds was screened using widely spread concentrations as described for that of metal testing. Definitive concentrations were then chosen. Experiments were carried out in 10% Bristol's medium with or without the addition of 0.4 m M Triton. The algal inoculation procedure was as before. The Triton-organics pairing effect on Chlorella cells was further investigated with hyamine at different Triton concentrations. To each hyamine dosage of 0.32, 0.80, 1.6 and 2.4 m g - 1 - ~ in 10% Bristol's, a sequence of Triton concentrations, i.e., 0.1, 0.2, 0.4, 0.8 and 1.2 mM, was added. Assay procedures were carried out as before. Chlorella biomass was measured by reading the optical density of the culture medium at the end of the incubation period. Results are presented as the mean. Relationships between treated and control samples were investigated using linear regression analysis while their differences were evaluated using the t-test for match pairs and the significance values reported at the P < 0 . 0 5 level. RESULTS
Routine assays with natural waters The water qualities of the study lakes are described in Table I. The remote lakes *Hyamine 3500, an 80°70 concentrate in ethanol, has an active ingredient n-alkyl (50% CI4, 40% C12, 10070 C~6) dimethylbenzylammoniumchloride.
Heart L.
Penetang Bay
Gravenhurst Bay
Urban lakes Aquitaine Reservoir
Trout L.
Swan L.
Ruth Roy L.
Plastic L.
Miskokway L.
McCann L.
Heeney L.
Chub L.
Remote lakes Bowland L.
Study lakewaters
43°35 ' N 79045 ' W 44°55'N 79°24'W 44°46'N 79o56'W 43°44'N 79047 ' W
47o05 ' N 80 °50' W 45 ° 13' N 78o59 ' W 45°08'N 79 ° 06' W 45°45'N 79 ° 10' W 45°39'N 80o14'W 45 ° 11' N 78o50'W 46°05'N 81o14'W 46o22 ' N 81o04'W 45°35 ' N 80 °04' W
Coordinates (Lat. (°N) Long. (°W))
relaorted by Suns et al. (1980).
7.6
8.0
7.1
8.1
6.2
5.1
4.8
6.2
6.0
6.3
5.9
5.5
5.4
pH
102.0
85.0
17.0
146.0
15.0
15.0
6.0
7.0
10.0
N/A
7.0
8.0
16.0
Hardness (CaCO3 mg • 1- l)
0.087
0.023
0.014
0.046
0.004
0.005
0.001
0"005
0.005
N/A
0.005
0.005
0.003
Total phosphorus (TP) (mg • 1- i)
1.11
0.33
0.34
0.65
0.20
0.23
0.12
0.31
0.35
N/A
0.24
0.28
0.20
Total Kjeldahl nitrogen (TKN) (mg • 1- i)
9.6
3.3
4.2
3.6
3.9
1.1
0.5
2.4
3.6
N/A
2.5
4.4
2.1
Dissolved organic carbon (DOC) (mg • 1- l)
Gravenhurst Bay PCB 350+ 85 ~ D D T 83 + 14 Dieldrin 5 + 2 Chlordane 3+_1 DDE
Chub L. PCB trace DDE trace
Heeney L. PCB 256 + 84 DDE 20
Organic residues in yearling yellow perch (ng• g - i wet wt.)
Some water quality characteristics of the 13 study lakes. The mean concentrations of organic residues analysed from the fatty tissues of the yearling perch
TABLE I
120
situated in the precambian Shield, about 200 km north of Toronto, are generally unproductive, with total phosphorus concentrations below 0.005 mg-1- 1 and total Kjeldahl nitrogen concentrations below 0.35 mg-1-1. These waters are low in CaCO3, between 6 and 22 m g - l - 1 and their pH often fails below 6.3. The urban lakes, on the contrary, are enriched waters with high nutrients (TP between 0.046 and 0.087 mg-I - 1 and TKN between 0.33 and 1.11 mg.l-1), high CaCO3 concentrations (between 85 and 146 mg. 1-1) and high pH values (7.6-8.1) except Graverhurst Bay which is more of a softwater urban lake with CaCO3 hardness of 17 mg.1The Chlorella growth curves observed from routine assays with these enriched, filtered lakewaters portray two kinds of algal response (Fig. 1). The first kind represented by Chub L. (a humic lake) and Stream A (entering Plastic L.) shows a normal pattern of Chlorella growth; the algal biomass increases during the early days of incubation to attain a logarithmic growth phase, then the growth slope gradually levels off to establish a maximum yield plateau when a certain limiting factor sets in. The second kind, denoted by the annihilation of Chlorella growth in the cultures as depicted by Miskokway L. and Heart L. in the figure, occurs rather frequently in our study in both the hard and soft waters. During incubation, Chlorella cells in these water media were quite insensitive to the addition of the chelator, NazEDTA; they formed clusters sometimes of up to a hundred ceils or more.
10 7 Chub Lake (humic water) Stream A (Plastic Lake) o Heart Lake [hardwater) • Miskokway Lake (softwater) []
106
T
E 03 J ,...J LL]
0 < J
k~ 105 122
0
I
0
104
I
I
I
I
I
I
I
2
4
6
8
10
12
14
DAYS
Fig. 1. Growth response of Chlorella cells in natural water media enriched with 10% Bristol's solution.
121
Organic contaminants were undetected in the water analysis. The detection limits for certain compounds such as PCB, DDT and chlordane for example are 20, 5 and 2 ng.l-a, respectively. The dissolved organic carbon concentrations (DOC) were also low, ranging between 0.5 and 4.4 mg.1-1 except for Heart L. (Table I). Although the organic trace contaminants had concentrations far below the detection limits from the water analysis, Suns et al. (1980) from their study of the bioaccumulation of organics in fish tissues, illustrated substantial quantities of PCB, DDT, chlordane and dieldrin embedded in the fatty tissues of yearling yellow perch from three of our study lakes (Table I). The PCB concentrations of between 256 and 350 ng.g-1 were the most prominent among the tested compounds in these waters. Response of Chlorella cells to Triton X-lO0 The growth of Chlorella cells did not show significant changes with the addition of Triton X-100 in 10°70 Bristol's medium (Fig. 2). The specific growth rates calculated were not significantly different among the treated samples and the con107
o
control
• 4- 0.2mM Triton X-100 v "1- 0.4raM Triton X-100 D-I-0.8mM Triton X-t00
E
106
o z
o I-< rr I-IZo
105
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0 0 ..J ..J LIJ
sp. gr. rate (d"1)
o
max yield (cells ml't)
T=I= S.E. 104
control
~'~S.E.
0.48---+0.01
2.0 x 1064"0.1 x 106
+0.2raM Triton X-100
0.54"+'0.05
2.3 x 106--+0.5 x 106
+0.4mM Triton X-100
0.53"1-0.13
1.5 x 1064"0.2 x 106~
-1"0.8rnM Triton X-100
0.48_--l_--~0,09
1.6 x 1064"0.3 x 106~
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t
I
t
I
I
t
2
4
6
8
10
12
14
DAYS Fig. 2. G r o w t h response o f Chlorella ceils in 10% Bristol's m e d i u m with and without Triton X-100 addition. *Indicates significant differences ( P < 0 . 0 5 ) f r o m the control; n = 3.
122
trol (inset, Fig. 2). Very slight drops in Chlorella yields were observed in Triton treated samples o f 0.4 and 0.8 raM. Although the decrease in biomass appeared to be negligible, there was a significant difference ( P < 0.05) between the sample means o f the control and the Triton treated cultures.
Pairing effect of Triton X-IO0 and metals on Chlorella cells Chemically, Triton produced a synergistic effect o f metal toxicity on Chlorella cells. When mixed with 0.4 mM Triton at low Cu 2+ concentrations, growth of Chlorella cells appeared to be stimulated; but at higher Cu 2 ÷ concentrations o f around 0.04 mg" 1- 1, Cu 2 + toxicity was enhanced by Triton with complete inhibition of Chlorella growth observed at about 0.065 mg Cu z + .1-1 (Fig. 3a). The synergistic effect was more obvious when Triton was paired with Cd 2+. Chlorella growth decreased appreciably in the Cd 2 + cultures when Triton was added (Fig. 3b). Since Cd 2÷ is not as detrimental as Cu 2+ (Wong and Beaver, 1981), it
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123 v p-chlorophenol-4- 0.4mM Triton X- 100 0.6 t
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FORMALDEHYDE (%) Fig. 4. Growth response of Chlorella cells to (a) p-chlorophenol, (b) pentachlorophenol, (c) herbicide (Crabgrass Killer FMC), and (d) formaldehyde with and without the addition of Triton X-100 in 10% Bristol's medium.
124
took 1.25 mg" 1- a of Cd z + (about 20 times the inhibit growth of Chlorella cells.
Cu 2 +
concentration) to completely
Pairing effect o f Triton X-IO0 and organics on Chlorella cells In contrast to its synergistic effect with metals, Triton when combined with organic compounds was found to have an ameliorating effect on Chlorella growth (Fig. 4). At 0.4 mM, Triton was capable of relieving part of the toxic stress on Chlorella cultures exposed to formaldehyde and the herbicide (Crabgrass Killer) (Fig. 4c, d). When coupled with pentachlorophenol and p-chlorophenol, Triton showed a more complete antagonistic reaction with these compounds thus allowing better growth of Chlorella cells in the assays (Fig. 4a, b). Triton's growth revitalizing ability with organics was most pronounced when paired with hyamine, an algicide. By using maximum algal yield as the measuring parameter, it was observed from a sequence of Triton concentrations that a better Chlorella yield ensued with every increase in concentration of Triton in the medium until the biomass finally reached its maximum yield plateau (Fig. 5a). Then from the biomass yield curves constructed in Fig. 5a, the binding concentration of Triton or the amount of Triton required to immobilize the hyamine activity 25 o 0.32 rng hyamine 1-1 a)
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Fig. 5. (a) Chlorella biomass curves constructed from yields obtained from various combinations of hyamine and Triton X-100 pairings. The ascending slope was extrapolated to intercept at the extended maximum yield plateau for the estimation of the binding concentration of Triton or the amount of Triton required to immobilize all organic activities in the medium. (b) The relationship between the binding concentration of Triton and the hyamine dosage used.
125
in the medium can be estimated by extrapolating the ascending slope of the biomass curve to intercept the line extended from the maximum yield plateau as demonstrated in the figure. Further, by plotting the binding concentration of Triton against the hyamine dosage used, a relationship between Triton and hyamine can be established to estimate the toxic stress of a test water sample in terms of hyamine dosage if the amount of Triton required to achieve a Chlorella maximum yield plateau is known (Fig. 5b). Evaluation of toxicity o f trace contaminants in waters Finally, when assays were tested with enriched natural waters treated with Triton, Chlorella cells that were previously growth inhibited (such as those in control cultures with zero Triton in Figs. 6 and 7) became activated and recovered steadily
REMOTE LAKES ~ B o w l a n d Lake 0.4
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T R I T O N X-100 (mM) Fig. 6. Chlorella b i o m a s s versus Triton X - I 0 0 for remote lakes. Z e r o Triton concentration was used as a control.
126 0.3 URBAN LAKES ~' Aquitaine Reservoir • GravenhurstBay o Heart Lake 0"3 .<
a PenetangBay
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TRITON X-100 [mM] Fig. 7. Chlorella biomass versus Triton X-100 for urban lakes. Zero Triton concentration was used as a control.
when accompanied by increasing Triton concentrations until the maximum yield plateau was attained. When the biomass curves o f the test waters were compared, T A B L E II The trace organic toxic stresses translated into mg h y a m i n e . l - ~ for our study lakes. Study lakewaters
Organic toxic stress (mg hyamine • 1-)
Estimated binding conc. of Triton (mM)
Remote lakes Bowland L. Chub L. Heeney L. M cCan n L. M i s k o k w a y L. Plastic L. Ruth Roy L. Swan L. Trout L.
0.7 0.0 0.8 0.8 0.8 0.5 2.3 1.5 0.8
0.40 0.00 0.50 0.45 0.48 0.24 1.00 0.80 0.46
Urban lakes Aquitaine Reservoir Gravenhurst Bay Penetang Bay Heart L.
1.0 1.5 1.0 0.7
0.62 0.80 0.60 0.40
127 the urban lakes showed markedly lower biomass plateaus than those of the remote lakes. Following the approach described earlier, the binding concentrations of Triton which permit maximum Chlorella yields were estimated. The binding concentration for Miskokway L., for example, as constructed by the dotted lines (Fig. 6) was estimated to be 0.5 mM Triton and according to the Triton-hyamine curve in Fig. 5b, the organic toxic stress was translated as 3.6 mg hyamine.lThe toxicity of trace organic contaminants for our study lakes is translated in Table II. The highest stress was obtained from Ruth Roy L. at about 2.3 mg hyamine.1-1 and the lowest was from Plastic L. at about 0.5 mg hyamine-1-1 These estimated organic toxic stresses, however, showed no correlation with the DOC measurements of the lakewaters. DISCUSSION Organic contaminants have been suspected in many lakes in southern Ontario when Chlorella assay cultures failed repeatedly with treatments of both nutrients and the chelator Na2EDTA. Somehow, because of extremely low levels, mostly in the ng' l- i range, contaminants such as PCB and DDT have not been perceived as significant growth inhibitors of biotic communities thus far. Only in more recent studies have scientists begun to realize through hioaccumulation studies of living tissues such as fish and clams, and from the analysis of sediments in urban and remote lakes, that many trace organic compounds such as polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB) and insecticides are, in fact, prevalent in aquatic environments and are a potential threat to many sensitive species (Hamelink et al., 1975; Addison, 1976; Butler and Schutzmann, 1977; Tan and Heit, 1981; Tsui and McCart, 1981; Gooch and Hamdy, 1983; Niimi, 1983). Contaminants in water are absorbed by fish and accumulated in their tissues. Suns et al. (1980) demonstrated magnified concentrations of PCB, DDT, chlordane and dieldrin residues in yearling perch from three of our study lakes. Perhaps the bioavailability of these trace organic substances helps to explain why growth of Chlorella cells was inhibited in assays with these waters and also why Chlorella cells formed clusters instead of forming enlarged, single deformed cells similar to those observed in tests with metals. Many surfactants, especially those in the octylphenol and nonylphenyl series, are effective binding substances for organic compounds in solutions besides serving as emulsifiers or surface active agents. At high concentrations, Triton can effectively retain organic residues by aggregating the hydrophobic portion of the molecules in groups and keep them inactive in the internal region of the micelles (Swisher, 1970; Abel, 1974). There are definitely a number of other organic polymers that have a great affinity for organic compounds (Gustafson and Paleos, 1971; Healy, 1971;
128 Dressier, 1979) but among our tested nonionic surfactant series of polyoxyethyleneglycol (Tween) and polyethoxyethanol (Triton), Triton X-100 appears to be the only effective agent which by itself exerts little harmful effect on Chlorella cells in the assays. At low concentrations, as reported in the literature, Triton has not been found to hamper the growth process of living cells except those of bacteria and virus cells mentioned by Simmons et al. (1977). Triton has no effect on the glucose oxidative process of human lymphocytes (Hrabak, 1982); it does not impede germination of Petunia pollen grains (Pfahler et al., 1981); it only slightly moderates Chlorella growth in our study. Above all, it is important that Triton does not serve as a growth stimulant (Fig. 2) or as a source of organic carbon to Chlorella cells. By solubilizing the protein and lipid layers in cell membranes as suggested by Capaldi (1977) and Ukekes (1965), Triton probably enhances large influxes of metal into the cells and thus magnifies their toxicity in the cultures. But chemically, Triton does not react with metals such as Cu 2 + in solution. During the polarographic calibration of Cu 2÷ when 0.01% of Triton was immersed in the metal solution, there was no significant change observed in the calibrated curve of Cu 2 + nor was there any apparent reduction in the height of the maximum, except the smoothing out of the curve which is generally regarded as a physical phenomenon. On the other hand, because of the micellar interaction, Triton restricts the mobility of organic compounds such as PCP, p-chlorophenol, formaldehyde and hyamine, and prevents them from interacting with the Chlorella cells such that when sufficient Triton is available in the medium, Chlorella cells, protected from the organic toxicants, are capable of attaining their maximum yield at the growth plateau (Fig. 5a). Chlorella cells responded favourably to enriched natural water media once Triton was introduced (Figs. 6 and 7). On the assumption that organic residues were present in trace quantities in these waters as evidenced by Suns et al. (1980) and also Triton could immobilize activities of organic materials, the recovery of Chlorella cultures in our assays suggests the predominance of organic toxicity in these waters. If metals were the controlling factor, the treatment of these waters with Triton would have only deepened the growth depression and certainly not revived the growth of Chlorella cells. Once the organic stresses in the water systems were quantitatively estimated, as shown in Table II, a better interpretation of the water quality of these waters could be made even just by comparing these data alone; for example, urban lakewaters suggested a higher burden of organic wastes while the remote lakes, on the whole, were less affected by organic pollutants except Swan L. and Ruth Roy L. which showed high toxicity values. Containing abundant suspended matter, Chub, the humic lake, was observed to be free from organic toxic stresses. It is obvious that there are other growth inhibitors, e.g., metal complexes, pH, etc., present in these waters, particularly those of the urban lakes which showed ex-
129
tremely low yields in the biomass plateaus (Fig. 7). Only by integrating this method with the approaches we suggested earlier on total toxicity and metal mixtures, may we perhaps develop a better understanding of the inorganic fraction of toxicity in these waters (Wong and Beaver, 1980; Wong, 1984). In conclusion, the bioaccumulation of organic residues in fish tissues suggests the bioavailability of trace contaminants that are not easily detectable in routine water analysis. Triton X-100, effectively reducing the toxic effect of single test compounds in Chlorella assays, presumably behave similarly to immobilize molecules of organic mixtures in natural waters. Despite its positive ameliorating growth effect with organic compounds on Chlorella, Triton could only mitigate a toxic effect no greater than an equivalent of about 2.4 mg hyamine • 1- 1 in the cultures. Biomonitoring, whether with fish or clams, is a good device to detect the presence of trace toxicants but it will be more valuable if the toxicity of the organic trace mixtures can be quantified at the same time. It is hoped that the bioassay approach suggested above will have some use in future assessment of organic toxicants in natural waters. REFERENCES Abel, P.D., 1974. Toxicity of synthetic detergents to fish and aquatic invertebrates. J. Fish. Biol. 6, 279-298. Addison, R.F., 1976. Organochlorine compounds in aquatic organisms: their distribution, transport and physiological significance. In: Effects of pollutants on aquatic organisms, edited by A.P.M. Lockwood, Cambridge Univ. Press, London, pp. 127-144. Alfheim, I., J. Gether and G. Lunde, 1978. Access of some atmospheric non-polar organic micropollutants to lakes and rivers. Acid precipitation - effects on forest and fish project. Res. Report 14, Norway. Anon., 1981. Triton surface-active agents. Rohm and Haas, Philadelphia, PA, 28 pp. American Public Health Association, 1980. American Water Works Association and Water Pollution Control Federation, Bioassay methods for aquatic organisms. In: Standard method for the examination of water and waste-water, 15th edition, edited by A.E. Greenberg, J.J. Connors and D. Jenkins, APHA, Washington, DC, 615 pp. Blaise, C., R. Legault and N. Bermingham, 1983. A simple microassay technique for measuring algal growth inhibition (ECs0's) in aquatic toxicity studies. Proceeding of the 9th annual aquatic toxicity workshop, Nov. 1-5, 1982. Edmonton, Alberta, edited by W.C. McKay, Canadian Technical Report of Fish. and Aquat. Sci. No. 1163, pp. 1-8. Butler, P.A. and R.L. Schutzmann, 1977. Bioaccumulation of DDT and PCB in tissue of marine fish. ln: Aquatic toxicology, edited by L.L. Marking and R.A. Kimerle, ASTM 667, Philadelphia, PA, pp. 212-220. Capaldi, R.A., 1977. The structural properties of membrane proteins. In: Membrane protein and their interactions with lipids, edited by R.A. Capaldi, Plenum, NY, pp. 1-20. Cooke, M. and A.J. Dennis, 1981. Chemical analysis and biological fate: polynuclear aromatic hydrocarbon, 5th Int. Syrup., Battelle Press, 770 pp. Cruickshank, J.H., 1968. Industrial uses of non-ionic surface-active agents. Water Pollut. Control. Symposium paper 5, pp. 100-107. Donaldson, W.T., 1977. Trace organics in water. Env. Sci. Technol. 11, 348-351. Dressier, M., 1979. Extraction of trace amounts of organic compounds from water with porous organic polymers. J. Chromatography 165, 167-206.
130 Ferrard, J.F., P. Vasseur and J.M. Jouany, 1983. Value of dynamic tests in acute ecotoxicity assessment in algae. Proceedings of the 9th annual aquatic toxicity workshop, Nov. 1-5, 1982. Edmonton, Alberta, edited by W.C. MacKay, Canadian Technical Report of Fish. and Aquat. Sci. No. 1163, pp. 38-56. Geyer, H., G. Politzki and D. Freitag, 1984. Prediction of ecotoxicological behaviour of chemicals: relationship between n-octanol/water partition coefficient and bioaccumulation of organic chemicals by alga Chlorella. Chemosphere 13, 269-284. Gooch, J.A. and M.K. Hamdy, 1983. Uptake and concentration factor of Aroclor 1254 in aquatic organisms. Bull. Environ. Contam. Toxicol. 31, 445-452. Gustafson, R.L. and J. Paleos, 1971. Interactions responsible for the selective adsorption of organics on organic surfaces. In: Organic compounds in aquatic environments, edited by S.J. Faust and J.V. Hunter, Marcel Dekker, Inc., NY, pp. 213-238. Hamelink, J.E., R.C. Waybrant and P.R. Yant, 1975. Mechanism of bioaccumulation of mercury and chlorinated hydrocarbon pesticides by fish in lentic ecosystems. In: Fate of pollutants in the air and water environments, edited by 1.H. Suffer, Wiley-lntersci., pp. 261-281. Healy, T.W., 1971. Selective adsorption of organics on inorganic surfaces. In: Organic compounds in aquatic environments, edited by S.J. Faust and J.V. Hunter, Marcel Dekker, Inc., NY, pp. 187-212. Hrabak, A., 1982. Damaging effect of detergents on human lymphocytes. Bull. Environ. Contain. Toxicol. 28, 504-511. Hutchinson, T.C. and P.M. Stokes, 1975. Heavy metal toxicity and algal bioassays. Amer. Soc. for testing and materials. Special Tech. Publ. 573, pp. 321-343. International Organization for Standardization (ISO), 1980. Algal growth inhibition test, draft method, Delft, The Netherlands. Joubert, G., 1980. A bioassay application for quantitative toxicity measurement using the green algae Selenastrum capricornutum. Water Res. 14, 1759-1763. Lillie, T.H., R.E. Hampson, Y.A. Nishioka and M.A. Hamilton, 1982. Effectiveness of detergent and detergent plus bleach for decontaminating pesticide applicator clothing. Bull. Environ. Contain. Toxicol. 29, 89-94. Lunde, G. and A. Bjorseth, 1977. Polycyclic aromatic hydrocarbons in long-range transported aerosols. Nature 268, 518-519. Metcalf, R.C., 1977. Biological fate and transformation of pollutants in water. In: Fate of pollutants in the air and water environments, edited by i.H. Suffet, Wiley-lnterscience, pp. 195-221. Niimi, A.J., 1983. Biological and toxicological effects of environmental contaminants in fish and their eggs. Can. J. Fish. Aquat. Sci. 40, 306-312. Organization for Economic Cooperation and Development (OECD), 1981. Guidelines for testing of chemicals, Paris, France, 201 pp. Payne, A.G. and R.H. Hall, 1979. A method for measuring algal toxicity and its application to the safety assessment of new chemicals. In: Aquatic toxicology, edited by L.L. Marking and R.A. Kimerle, ASTM 667, Philadelphia, PA, pp. 171-180. Pfahler, P.L., H.F. Linskens, H.W. Schoot and M. Wilcox, 1981. Surfactant effects on Petunia pollen germination in vitro. Bull. Environ. Contam. Toxicol. 26, 567-570. Schauberger, C.W. and R.B. Wildman, 1977. Accumulation of aldrin and dieldrin by blue-green algae and related effects on photosynthetic pigments. Bull. Environ. Contam. Toxicol. 17, 483-488. Simmons, K., H. Garoff and A. Helenius, 1977. The glycoproteins of the semliki forest virus membrane. In: Membrane proteins and their interactions with lipids, edited by R.A. Capaldi, Plenum, NY, pp. 207-234. Starr, R.C., 1,964~ The culture collection of algae at Indiana University. Amer. J. Bot. 51, 1013-1044. Suns, K., C. Curry and D. Wilkins, 1980. The effects of road oiling on PCB accumulation in aquatic life. Ontario Min. of Environ., 14 pp. Swisher, R.D., 1970. Surfactant biodegradation. Marcel Dekker, Inc., NY, 496 pp. Tan, Y.L. and M. Heit, 1981. Analysis of polynuclear aromatic hydrocarbons in sediment cores from
131 two remote Adirondack lakes by resolution gas chromatography/mass spectrometry. In: Polynuclear aromatic hydrocarbons. 5th Int. Symp., Battelle Press, pp. 561-570. Tsui, P.T.P. and P.J. McCart, 1981. Chlorinated hydrocarbon residues and heavy metals in several fish species from the Cold Lake area in Alberta, Canada. Inter. J. Env. Anal. Chem. 10, 277-285. Ukeles, R., 1965. Inhibition of unicellular algae by synthetic surface active agents. J. Pt~ycol. 1,102-110. U.S. Environmental Protection Agency, 1978. The Selenastrum capricornutum Printz. algal assay bottle test: experimental design, application and data interpretation protocol. USEPA, Environmental Research Laboratory, Corvallis, OR, 126 pp. Wong, S.L., 1984. Toxicity and water quality: a bioassay interpretation. J. Env. Sci. Health 19, 377-386. Wong, S.L. and J.L. Beaver, 1980. Algal bioassays to determine toxicity of metal mixtures. Hydrobiol. 74, 199-208. Wong, S.L. and J.L. Beaver, 1981. Metal interactions in algal toxicology: conventional versus in vivo tests. Hydrobiol. 85, 67-'/1. Wong, P.T.S., Y.K. Chau, O. Kramar and G.A. Bengert, 1982. Structure-toxicity relationship of tin compounds on algae. Can. J. Fish. Aquat. Sci. 39, 483-488. Zoetman, B.C.J., K. Harmsen, J.B.H.J. Linders, C.R.H. Morra and W. Slooff, 1980. Persistant organic pollutants in river water and ground water of the Netherlands. Chemosphere, 9, 231-249.