Structure-toxicity of triaryl phosphates in freshwater algae

The Science o f the Total Environment, 32 (1984) 157--165 Elsevier Science Publishers B.V., Printed in The Netherlands

157

STRUCTURE-TOXICITY OF T R I A R Y L PHOSPHATES IN F R E S H W A T E R ALGAE

P.T.S. WONG 1 and Y.K. CHAU 2 Fisheries and Oceans, Great Lakes Fisheries Research Branch 1, National Water Research Institute 2, Canada Centre for Inland Waters, Burlington, Ontario L 7R 4A6 (Canada) (Received March 16th, 1983; accepted April 20th, 1983)

ABSTRACT The effects of six triaryl phosphate compounds on the primary productivity of pure algal cultures and natural phytoplankton in lake water were tested. Substitution of the hydrogen atom by a methyl group in the benzene ring decreased its effect. Triphenyl phosphate was the most toxic compound, followed by tolyl diphenyl phosphate, tritolyl phosphate and trixylyl phosphate. The ortho-isomer of tritolyl phosphate was more toxic than the meta- and para- isomers. Natural phytoplankton was more sensitive to triaryl phosphate compounds than the pure algal cultures. In addition to the primary productivity, the reproduction and nitrogenase activity of the algae were also reduced in the presence of triphenyl phosphate.

INTRODUCTION

Triaryl phosphates (TAP) are organophosphate esters. They are widely used in many consumer and industrial products such as in plastics and hydraulic fluids (Holliday et al., 1982). Much of these compounds may be eventually released into the environment either by direct release to air and water, or from land disposal (Midwest Research Institute, 1979). Many of these c o m p o u n d s have been found in water (LeBel et al., 1981), sediments (Mayer et al., 1981) and fish (Lombardo and Egry, 1979). Limited information of the toxicity of TAP to aquatic organisms is available. The acute toxicity of pure and mixed TAP toward fish ranged from a high 96 h LCs0 value of 100 mg/1 (Nevins and Johnson, 1978) to a low value of 0.31 mg/1 (Sitthichaikasem, 1978). Salmonids were generally more sensitive to TAP than fathead minnows and the age of the rainbow t r o u t did not affect sensitivity (Mayer et al., 1981). Sublethal effects of these c o m p o u n d s on fish included morphological, behavioural and biochemical abnormalities (Wagemann et al., 1974; Lockhart et al., 1975). Invertebrates were reported to be less sensitive to TAP than fish (Nevins and Johnson, 1978; Mayer et al., 1981; Holliday et al., 1982). Not much is known a b o u t the toxicity of TAP to algae. One report showed that a green 0048-9697/84/$03.00

© 1984 Elsevier Science Publishers B.V.

158 alga, Selenastrum capricornutum, was very resistant to a commercial mixture of TAP c o m p o u n d s (Mayer et al., 1981). No other information is available. In this report, six commercially important TAP compounds were tested for their effects on the primary productivity of three pure algal cultures as well as natural p h y t o p l a n k t o n from Lake Ontario. The reproduction of Ankistrodesmus falcatus and the nitrogenase activity o f Anabaena flos-aquae in the presence of TAP compounds were also studied. These compounds were chosen because of the presence of the methyl-group substitution in the phenyl rings. The possible relationship between the toxicity and the methylgroup substitution of the compounds in the algae was examined.

MATERIALS AND METHODS

Cultures Three algal cultures were used: Scenedesmus quadricauda (Culture collection No. 11, Dr. P. Healey, Freshwater Institute, Winnipeg, Manitoba), Ankistrodesmus falcatus variant acicularis and Anabaena flos-aquae (Ontario Ministry of Natural Resources, P.O. Box 213, Rexdale, Ontario). All cultures, except Anabaena flos-aquae, were maintained in a modified CHU-10 medium at pH 8 (Wong et al., 1978). Anabaena flos-aquae was maintained in a modified nitrogen-free BG-11 medium (Stratton and Corke, 1979). The inoculum for toxicity bioassays was prepared by growing each culture in 100 ml of medium at 20°C on a rotary shaker (100 rpm) under conditions of 1 8 h of light (50001ux) and 6 h of darkness. When the cells reached the logarithmic phase of growth (about 1 week), they were used as inoculum.

Primary productivity experiments Primary productivity was measured by the amount of [14C] carbonate taken up by algae over a 4-h period. Of the culture 1 ml (7 X l 0 s cells/ml) was added to 13.9 ml of CHU-10 medium containing various concentrations (0--5mg/1) of TAP compounds dissolved in acetone (see under section Chemicals) in a 25-ml erlenmeyer flask. Equivalent volumes (5 #1) of acetone were used as controls. Acetone had no effect on primary productivity if the volume used was less than 0.05% of the medium. After a 24-h incubation at 20°C under conditions described above, a 0.1-ml aliquot of 7.4 x 104 Bq/ml sodium [14 C] carbonate (Amersham/Searle, 2.2 × 109 Bq/mmol) was added to each flask. The flasks were tightly capped with rubber stoppers wrapped in aluminum foil. A similar set of flasks was incubated in the dark. After a further 4-h incubation, the cells were fixed with 0 . 0 5 m l of neutralized formalin. They were filtered through a 0.45-#m membrane filter and rinsed rapidly with 10-ml fresh CHU-10 medium to remove extracellular sodium [14 C] carbonate. Filters containing radioactive labeled cells were dissolved in 10 ml PCS scintillation counting fluor (Amersham/Searle). Radioactivity was measured by liquid scintillation counter with an automatic quenching factor and a 10,000 dpm upper limit. Radioactivity taken up by algae in the dark

159 ~ 3

0

OH3

o\// p

~[[[~-o\//o

,o©

CH3 CH3 TRI- ORTHO-TOLYL-PO4(TOTP) (TRI-ORTHO-CRESYL-P04)

CH3 =j

P

0\//, 0

OH3 CH3 TRI- META-TOLYL- P04(TMTP) (TRI-META-CRESYL-P04) /~-x CH3 GH3~X

p

0 \// 0 P

TRI-PARA-TOLYL P04 (TPTP) (TRI-PARA-CRESYL-P04)

OH3 OH3 TRI-2,4,- DIMETHYLPHENYLP04 (TRI-XYLYL- PO4)(TXP)

o

p

TRIPHENYL-P04(TPP)

P

TOLYL DIPHENYLPO4(TDP) (CRESYL DIPHENYL-POa)

Fig. 1. The structural formulae of six triaryl phosphates. ( < 5% of total) was subtracted from the total radioactive counts. Radioactivity in the algae not exposed to TAP compounds (control) was taken as 100%. The concentration causing a 50% reduction in primary productivity and reproduction (median inhibition concentration, ICs0 ) was calculated by probit analysis (Finney, 1971). Toxicity of TAP c o m p o u n d s on a natural algal c o m m u n i t y was tested with water from a sampling site in Lake Ontario near Hamilton, Ontario, Canada. To compensate for the smaller number of algae, the volume of water was increased from 15 to 100 ml. Of 3.7 x l 0 s Bq/ml sodium [ 14 C] carbonate 150 ml was used. Other conditions were the same as in culture experiments.

Algal reproduction experiment The effects of TAP c o m p o u n d s on reproduction (growth) of A. falcatus were measured spectrophotometrically with a Klett-Summerson Photoelectric colorimeter. Of the inoculum 1 ml was added to 49 ml CHU-10 medium with and without TAP c o m p o u n d s in a 300-ml nephelco culture flask with side arm (Bellco Co., Vineland, NJ). At various intervals during incubation, the growth medium was tilted into the side arm which was then inserted into the colorimeter (with red filter No. 66). Because no medium is withdrawn from the flask for determination, this technique was more convenient and less prone to contamination.

160

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0.01

0~35

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CONCENTRATION ( mg/L )

Fig. 2. Effects of TAP compounds on the primary productivity of A. falcatus. Data presented as the mean of duplicate samples.

are

Nitrogenase activity Nitrogenase activity was measured with the acetylene reduction technique of Stratton and Corke (1979) with some modifications. Of Anabaena flosaquae (1 x 10 cells/ml) 9 m l were added to a 50-ml hypo-vial (Chromatographic Specialties Ltd., Brockville, Ontario). Of BG-11 medium 1 ml with and without triphenyl phosphate (TPP) was added. The vial was sealed with a tight-fitting rubber stopper and a 10% volume of air was removed and replaced with acetylene. In another experiment, cells were exposed to TPP for 24 h before acetylene was added. Activity was measured in triplicates at the end of 4 h of incubation at 20°C and a light intensity of 5000 lux. Of the gas sample 500 ml was removed from the sealed vial by a gas-tight syringe and injected into a Hewlett Packard model 5830 Gas Chromatograph, equipped with a hydrogen flame ionization detector and a Porapak N column (50--80 mesh, 10' x 0.125" O.D., stainless steel). The chromatograph was operated under the following conditions: detector, 200°C; injector, 150°C; column, 60°C; hydrogen 30 ml/min; air, 300 ml/min; and nitrogen, 53 ml/min. Acetylene and ethylene peaks were identified by retention time and quantitated by comparing peak heights to standard curves. Chemicals The six triaryl phosphate compounds with their structural formulae and abbreviations are shown in Fig. 1. These pure quality c o m p o u n d s were from Sargent-Welch Scientific Co. (Toronto, Canada) except TXP and TDP which were from Ventron Corporation (MA, U.S.A.). Since these c o m p o u n d s are not very soluble in water (Saeger et al., 1979) the stock solutions were

161 TABLE 1 SUMMARY OF ICs0 OF TRIARYL PHOSPHATE COMPOUNDS ON THE PRIMARY PRODUCTIVITY OF PURE ALGAE AND NATURAL PHYTOPLANKTON FROM LAKE ONTARIO WATER Medium inhibition concentration (ICs0) in mg/1. Compounds

TPTP TXP TMTP TOTP TDP TPP

A. falcatus S. quadricauda

~ 5.00 ~ 5.00 ~ 5.00 2.50 0.70 0.26

~ 5.00 _b ~ 5.00 4.20 1.00 0.50

Lake Ontario Water phytoplankton solubility

~ 5.00 -4.10 1.70 0.50 0.20

Octanol/water partition

(rag/l) a

coeff. (x 103) a

0.36 0.89 0.36 0.36 2.60 1.90

128 427 128 128 32 42

aSaeger et al., 1979 bnot determined p r e p a r e d in a c e t o n e and a d d e d t o t h e test m e d i u m to a final c O n c e n t r a t i o n o f 0--5 mg o f the c o m p o u n d per litre o f t h e m e d i u m .

RESULTS

Effect on primary productivity T h e r e s p o n s e o f A. falcatus t o various T A P c o m p o u n d s is s h o w n in Fig. 2. T P T P and T X P had little e f f e c t o n t h e algal p r i m a r y p r o d u c t i o n even at conc e n t r a t i o n s as high as 5 mg/1. O t h e r TAP c o m p o u n d s were i n h i b i t o r y . TPP was the m o s t t o x i c , f o l l o w e d b y TDP, T O T P and TMTP. S. quadricauda and a natural p h y t o p l a n k t o n f r o m L a k e O n t a r i o w a t e r were similarly a f f e c t e d b y these T A P c o m p o u n d s . C o n c e n t r a t i o n s o f T A P c o m p o u n d s c a u s i n g a 50% i n h i b i t i o n (ICso) in p r i m a r y p r o d u c t i o n o f p u r e and indigenous algae were e s t i m a t e d and s u m m a r i z e d in Table 1. These ICs0 values were useful for q u i c k c o m p a r i s o n o f toxicities. T h e ICs0 values o f TPP were 0.26, 0.50 and 0.20 mg/1 f o r A. falcatus, S. quadricauda and natural p h y t o p l a n k t o n f r o m Lake O n t a r i o respectively. T h e natural p h y t o p l a n k t o n f r o m L a k e O n t a r i o was the m o s t sensitive, f o l l o w e d b y A. falcatus, to TAP c o m p o u n d s , while S. quadricauda was m o r e t o l e r a n t . Within t r i t o l y l p h o s p h a t e c o m p o u n d s , the ortho- c o n f i g u r a t i o n o f t h e m e t h y l g r o u p was m o r e t o x i c t h a n the meta- and para-positions. No d i r e c t relationship b e t w e e n toxicities, w a t e r solubilities and o c t a n o l / w a t e r p a r t i t i o n c o e f f i c i e n t s o f T A P c o m p o u n d was observed.

Effect on reproduction Since T P P was the m o s t t o x i c T A P c o m p o u n d in r e d u c i n g the p r i m a r y p r o d u c t i v i t y o f algae, its e f f e c t o n the r e p r o d u c t i o n o f A. falcatus was

162 140v_..--------*--~v •

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/

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Days of Incubation Fig. 3. Effects o f ~ P o n the r e p r o d u c t i o n ( g r o w t h ) o f A. falcatus. Data are presented as the m e a n o f duplicate samples.

further examined. The results in Fig. 3 indicate the TPP at concentrations less than 0.1 mg/l did not reduce the reproduction of the alga. Instead it was even stimulating the reproduction as compared with the control algae (no TPP exposure). A t 0.5 mg/l of TPP, both the rate and the total amount of reproduction were decreased. Further increase in TPP concentrations to 1.0 mg/l and greater completely inhibited the algal reproduction.

Effect on nitrogenase activity In addition to the primary productivity and reproduction, the nitrogenase activity as measured by the acetylene reduction technique was also affected by TPP (Table 2). Addition of 0.1, 1.0 and 5.0 mg/1 of TPP reduced the nitrogenase activity of A. flos-aquae to 84, 77 and 68% respectively. Algae pre-incubated for 24h with the chemical did not exhibit an increase in sensitivity or resistance to the TPP toxicity.

DISCUSSION

Triphenyl phosphate (TPP) was found to be the most toxic TAP compound examined in this study (Fig. 2 and Table 1). It was also the most acutely toxic compound to fish, shrimp, daphnids and other algae (Midwest Research Institute, 1979; Mayer et al., 1981). Since TPP is a major component in several commercial TAP products (Midwest Research Institute, 1979) and it has been detected in various environmental samples (Lombardo and Egry, 1979; LeBel et al., 1981), its acute toxicity certainly warrants particular attention in environmental research. The presence of a methyl

163

TABLE 2 THE EFFECT OF TRIPHENYL PHOSPHATE (TPP) ON ACETYLENE REDUCTION BY A N A B A E N A FLOS-AQUAE (A) Cells not pre-incubated and (B) cells pre-incubated with TPP for 24 h before adding the substrate of acetylene.

Control 0.1 rag/1 TPP 1.0 mg/1 TPP 5.0 rag/1 TPP

(A)

(B)

0.62 + 0.01 a (100%) 0.52 + 0.04 (84%)

0.62 0.55 0.47 0.38

0.47 + 0.01 (77%) 0.42 + 0.02 (68%)

+ 0.01 +- 0.02 + 0.05 + 0.03

(100%) (89%) (77%) (61%)

a A m o u n t of ethylene (nmole) produced/h/10 s cells. Mean and standard deviation of three samples. Percentages in parenthesis show percentage of suppression compared to control (no TPP added).

group in the benzene ring, however, decreased its toxicity (Table 1). For example, TXP with two m e t h y l groups per benzene was relatively non-toxic as compared with tritolyl phosphate (one methyl group per ring) and TDP (one methyl group). No similar study for other aquatic organisms could be found for comparison. However, TPP was much more toxic that TOTP to fish (Holliday et al., 1982) and to guinea pigs, with the order of decreasing toxicity being TOTP > TDP > TXP (Dvorkin, 1973). In our results, no discemable relationship between the toxicities of these compounds and their physical properties (solubilities in water and octanol/water partition coefficients) was observed (Table 1). Therefore no obvious explanation for the differences in toxicity was available. However, it may be speculated that the position and the number of methyl-group substitution in the benzene ring could affect the transport and accumulation and consequently the toxicity of these c o m p o u n d s to the algae. For tritolyl phosphate compounds, the ortho-isomer was more toxic than the meta- and para-isomers (Fig. 2 and Table 1). A similar observation was reported for animals (Smith and Spalding, 1959). The higher toxicity of ortho-substitution was due to the ease of alkyl group to form a cyclic neurotoxic c o m p o u n d (Henschler, 1958). On the other hand, para-substituted nitrophenols were more toxic than the respective ortho-isomers while the latter were more avoided by fish (Zitko, 1976). A natural p h y t o p l a n k t o n from Lake Ontario was more sensitive to TAP than the pure algal cultures (Table 1). Similarly, natural algal populations were also more sensitive to organo--tin c o m p o u n d s than were the pure algal cultures (Wong et al., 1982a). The differences in sensitivity were attributed to differential sensitivity of the algal species (Wong et al., 1978), as well as to the different algal sizes (Munawar and Munawar, 1982). The sensitivity of fish species a l s o varied, with rainbow trout being the most sensitive species (Holliday et al., 1982). The route of administration of TAP to fish is very important on the severity o f toxicity effects. TPP was lethal to rainbow trout at concentrations as low as 1.4 pg/1 when exposed to the fish in water as compared with more than 1000 pg/g in f o o d (Holliday et al., 1982).

164 Apart from the primary productivity, other physiological indicators of the algal cells were also affected by TAP compounds. The reproduction of A. falcatus was reduced by exposure to 0.5 mg/1 of TPP (Fig. 3). At higher concentrations (1, 3 and 5mg/1) of TPP, growth was completely inhibited. Interestingly, at the lower concentrations of 0.1 and 0.05 mg/1 of TPP, the c o m p o u n d stimulated the growth. With bacteria, the TAP compounds could be used as carbon and phosphorus sources for growth (Pickard et al., 1975). A similar mechanism could possibly explain the stimulation of TPP in the algae. The genus Anabaena is a common freshwater blue-green alga and is responsible for much of the nitrogen fixation in freshwater lakes (Fogg and Stewart, 1965). Our results indicated that the nitrogenase activity of this alga was also sensitive to the TPP (Table 2). The nitrogenase activity of Anabaena sp. was also found less sensitive to metal inhibition than photosynthesis because the latter required the orderly operation of m a n y enzymes which were more likely to be exposed to metal inhibition than a single enzyme system in the nitrogenase activity (Wong et al., 1982b). In conclusion, the toxicities of TAP compounds to the freshwater algae depended on the structure o f the compounds. Substitution of the hydrogen ion by a methyl group in the benzene ring decreased its toxicities.

ACKNOWLEDGEMENT We thank Mrs. D. Patel for her excellent technical assistance and Caryl Fawcett for typing the manuscript.

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