tested. T h e company has developed a biological method for destroying harmful organic compounds contained in a particular waste solution. T h e active microorganisms involved are normally fed by addition of urea or ammonium nitrate. I t was found that these organisms thrived equally well on a diet of treated washwater containing some dinol. However, the output of nitrate from this source proved much too large to be absorbed by this process alone. I t has now been decided to work u p the dinol-extracted wash solution t o produce nitrate of commercial quality. This may be done by evaporation, preferably following recirculation of t h e wash solution (after adding more alkali), thereby increasing the nitrate concentration. Another way to go is ex-
traction of the nitrate according to a method developed in Finland ( 4 ) .In either case the toluene extraction step mentioned earlier will become a necessary part of the purification scheme. Literature Cited (1) a s t e r n . S., Norwegian Patent 134 885 (1976). ( 2 ) a s t e r n , S., U.S. Patent 4 047 989 (19771. ( 3 ) Pristera, F., A n a / . C h e n . , 25, 844 (1953). ( 4 ) Mattila, T. K.. Lehto. T. K.. I n d . !,'rig C'hem.,Process Des. Deu., 16,469 (1977).
Kccciicd f o r repieit, March 17, 1978. .4cc(,ptPd Januar?, 2. 1979
Biosynthesis and Release of Organoarsenic Compounds by Marine Algae Meinrat 0. Andreae" and David Klumpp2 Scripps Institution of Oceanography, University of California, San Diego, La Jolla, Calif. 92093 T h e uptake of arsenate from seawater, the biosynthesis of organoarsenic compounds, and the release of arsenite, methyl arsonate, and dimethyl arsinate have been studied in pure cultures of marine phytoplankton species, most of them bacteria free. Complex uptake kinetics and a wide variation in t h e degree of arsenic incorporation from the environment were found. In addition to a substantial amount of arsenic strongly bound to structural parts of the cell, u p to 12 soluble organoarsenic compounds were formed by the algae. All species released substantial amounts of methyl arsonate and dimethyl arsinate into their environment. The production of arsenite was also common, and especially conspicuous with two species of coccolithophores. These findings explain a t least in part the common occurrence of these arsenic compounds in the aquatic environment. Recent studies on the speciation of arsenic in the aquatic environment showed the occurrence of arsenite and two methylated forms of arsenic, monomethyl arsonate and dimethyl arsinate, in both terrestrial and marine waters (I-3j. In t h e marine environment, a strong positive correlation between the concentration of these forms and photosynthetic activity was observed: the methylarsenicals were found only in the euphotic zone, and their concentrations show a strong positive correlation with COe-assimilation rates. This suggested t h a t planktonic algae might be the producers of these compounds. Lunde ( 4 ) and Irgolic et al. (5) had shown that algae were able to take up arsenic from their environment and synthesize various water- and lipid-soluble compounds, which, however, remain to be chemically identified. Lunde ( 4j did not find methyl arsonate and dimethyl arsinate in the cell extracts by thin-layer chromatography. Neither Lunde nor Irgolic indicated t h a t their cultures were bacteria-free. Methods Bacteria-free algal cultures were obtained from the culture collection of the Food Chain Research Group of the Institute Present address, Department of Oceanography, T h e Florida State University, Tallahassee, Fla. 32306. * Present address, DeDartment of Botanv and Biochemistrv. Westfield College (Univkrsity of London), "Kidderpore Avenue, London NW3 7ST, U.K. 738
Environmental Science & Technology
of Marine Resources, University of California, San Diego. They were grown on IMR seawater medium (6). For the arsenate uptake experiments, 10-mL samples containing a total of 2.5 X IO6 cells were transferred into flasks containing 50 mL of medium with varying phosphate and arsenate concentrations. T h e background arsenate concentration of the medium was 13 nM. Carrier-free i4As tracer (2.2 X lo6 cpmj in the form of arsenate was added to the flasks. After the incubation the cells were filtered on Whatman GF/C or 1.2-pm Millipore filters and washed with tracer-free seawater, and the activity was determined in a well-type y counter. For the studies on the biosynthesis of organoarsenic compounds, the cells were grown in batch cultures of 60 mL in IMR medium. Initial concentrations of phosphate and arsenate were 25 p M and 13 nM, respectively. The cells were harvested by filtering on Whatman GF/C filters, except for Gonyaulax (the only culture that was not bacteria-free),which was filtered on an 8-pm Millipore filter. T o avoid cell disruption, the pressure differential during filtering was kept below 0.15 atm. The cells were then homogenized in a Virtis blender with a mixture of water. methanol, and chloroform (1:2:4),the phases separated, and their activities determined by y counting. Paper chromatography of the methanollwater extracts and the deacylated lipids was performed on SVhatman No. 4 paper. T h e extracts were developed with butanollwater (1800:121) mixed with an equal volume of propionic a c i d h a t e r (800: 1020). For thin-layer chromatography, a solvent system conwas used. The sisting of chloroform/methanol/water/NH~OH arsenic compounds were detected by autoradiography. The concentration of arsenite, arsenate. and the methylated forms of arsenic in natural waters and growth media was determined by the method of Andreae ( 7 ) ,which allows their detection in the parts per trillion range with a precision between 2 and 5?& The procedure is based on volatilization of the compounds by reduction with sodium borohydride, gas chromatographic separation of the hydrides, and their detection by atomic absorption and electron capture for the inorganic and organic species, respectively. Results a n d Discussion T h e chemical similarity between phosphate and arsenate suggests that these ions would show competitive behavior with
0013-936X/79/0913-0738$01.00/0
@ 1979 American Chemical Society
1,
~
10
20
30
40
50
60
minutes Figure 1. Short-term uptake of [i4As]arsenate in Platymonas cf. suecica from seawater (12.6 nM arsenate, 1.30 pM phosphate) I
011
I
0
regard to the phosphate uptake system. This has indeed been observed with Rhodotorula rubra, a marine yeast (6).This is of great importance in the open ocean, where arsenate and phosphate are often present in comparable concentrations. We studied the arsenate uptake by a bacteria-free culture of Platymonas cf. suecica using 74Asas a radioactive tracer. The amount of arsenic incorporated as a function of time is shown in Figure 1. An initial phase of very rapid uptake is followed by partial release of arsenic and then a slower, linear increase of cellular arsenic content. Arsenic uptake was followed over an interval of 5 days during which the cells were in exponential growth. T h e cellular arsenic content as estimated by the arsenic/chlorophyll ratio of harvested cells increased during the first 2 days, then stabilized while arsenic in the medium dropped by 35% from 13 nM and phosphate fell from 19 to 0.7 pM (Figure 2). Arsenate uptake was a linear function of arsenate concentration in the medium in the concentration range between 13 and 500 nM. Controls with cells killed by heating to 95 "C for 10 min showed no measurable uptake. The dependence of arsenate uptake on phosphate concentration did not show a simple competitively inhibitory pattern. Arsenate uptake increased when the phosphate concentration was raised from 0.4 to 2.4 p M , and started to decrease significantly only when phosphate reached 100 pM. These findings corroborate the apparent independence of cellular arsenic from environmental phosphate found in the 5-day experiment. A second long-term experiment was conducted with Cricosphera carteri, a coccolithophore. This species grew to stationary phase in 14 days without taking up enough arsenic to be detectable in aliquots of ca. 2 X IO6 cells. When the culture (1.5L)was finally harvested and the cells extracted, most of the arsenic they did contain was found to be insoluble in water/methanol/chloroform and apparently attached t o structural parts of the cell (Table I). However, 29% of the arsenate supplied in the media had been converted to arsenite. T h e complex short-term uptake behavior suggests t h a t arsenate is taken up by more than one mechanism. A simple competitive inhibition kinetics like in Rhodotorula rubra was not evident in the algal species studied. On the contrary, even
2
4
3
days
Figure 2. Uptake of arsenate in a batch culture of Platymonas cf. suecica. IMR medium was used without addition of unlabeled arsenic; the initial phosphate and arsenate concentrations were 19 pM and 13 nM, respectively. 2.7 X lo7 cpm of carrier-free [74As]ar~enate was added
large variations in phosphate concentrations, like the ones occurring in the batch cultures, did not significantly influence cellular arsenic concentrations. However, the behavior a t phosphate concentrations below 0.1 pm, which do occur in some parts of the open ocean, has not yet been adequately studied. Studies on the biosynthetic products formed were conducted on four species: Sheletonema co.qtatum, Platymonas cf. suecica, Gonyaulax polyedra, and Cricosphaera carteri. With the exception of Cricosphaera, all cultures incorporated significant amounts of arsenic (Table I). A substantial amount was insoluble in the solvent mixture, and could be extracted only with dilute HC1 or hot water. Paper chromatography of the water/methanol soluble fraction and thin-layer chromatography of the chloroform fraction showed four to six compounds in each phase (Table I1 and Figure 3). Deacylation of the Platymonas lipid fraction with 0.1 N KOH gave two compounds ( R f 0.18 and 0.41) in addition to any remaining lipid-soluble compounds which travel with the front in the propionic acid system. The products dissolved with hot water from the residue gave the same compounds as in the water/ methanol extract with the exception of the spot at R f 0.30, but in very different proportions. Arsenobetaine ((CH&As+CH2COO-] has been isolated from Panulirus longipes, the Australian rock lobster ( 8 ) ,and it has been suspected that it or arsenocholine [(CH3):jAs+CH2CH?OH] was an important compound in the phytoplankton extracts ( 5 ) .None of the compounds from the algal extracts, however, cochromatographed with arsenobetaine ( R f 0.701, and the substance which cochromatographed with arsenocholine ( R f 0.76) was separated from it by low voltage paper electrophoresis (acetic/formic acid, p H 2 ) . No signifi-
Table 1. Distribution of Arsenic between Insoluble Residue, Water/Methanol-, and Chloroform-Soluble Phase species
incorpa
total actlvlty, lo6 cpm
Platymonas cf. suecica Skeletonema costatum Gonyaulax polyedra Cricosphaera carterae
7a 44 13 4.6
0.11 0.23 0.15 0.11
Yo
a
% insoluble
water/ methanol
43 22 19 96
46 33 3.4
a
chiorof.
49 32 4a 0.6
The total amount of label incorporated in the plants (cpm of 74As on filter/cpm of 74As added to culture X 100).
Volume 13, Number 6, June 1979
739
Table II. Paper Chromatography of the Water/ Methanol-Soluble Fraction of Algal Extracts, the Deacylated Lipid Fraction of Platymonas cf. suecica (Platy. Deac.) and a Water Extract of the Residue of the Water/Methanol/Chloroform Extraction of P. suecica (Plafy, Residue) Rf values Skeletonema cosfafum
Gonyaulax polyedra
Plafymonar cf. sueclca
Plafy. deac.
Plafy. residue
0.22+++ 0.30+
0.22+++
0.18++
0.21+++
0.41-
0.53++
0.57' 0.74" 0.91-
0.16+++ 0.30++ 0.43+ 0.56+ 0.78"' 0.91-
0.43+ 0.52++ 0.75+
ii
0.98++
The symbols behind the R, values are an estimate of the relative abundances of the compounds. a
cant amounts of arsenate or arsenite could be detected in the extracts by autoradiography. However, some arsenate, methyl arsonate, and dimethyl arsinate were found by t h e more sensitive sodium borohydride reduction method. The amounts of the methylarsenicals in t h e water extracts of algal species can be significantly increased by heating with concentrated base. In addition, trimethylamine oxide is formed in these extracts. This suggests that methylarsenic configurations like those in arsenobetaine and arsenocholine are indeed found a t least in some of the compounds. T h e water-soluble organoarsenic compounds are very stable with respect t o concentrated acids. In some instances, organoarsenicals were still present in algal digests after several hours of boiling with a nitric/perchloric acid mixture. The release of organoarsenicals and arsenite was studied by filtering growth media from a number of plankton species and subjecting the filtrate to analysis (9)for arsenite, arsenate, methyl arsonate, and dimethyl arsinate. T h e results are summarized in Table 111, together with data for t h e uninoculated growth medium, surface seawater from a station off San Diego, and a water sample from the Salton Sea, a saline lake in Southern California that showed a strong dinoflagellate bloom, with a cell density comparable to laboratory cultures. All cultures showed a significant depletion of arsenic in the medium, except for Goniodoma cf. depressum, where very
h SC
GP
PS
P De Ac
Figure 3. Thin-layer chromatography of chloroform extracts of marine algae and the deacylation products of the chloroform extract of Platymonas suecica: SC, Skeletonema costatum; GP, Gonyaulax polyedra; PS, Platymonas cf. suecica; PDeAc, deacylated extract of P. suecica
little growth was evident, and Cricosphera carteri ZZ, where some contamination with arsenate may have occurred. All species also show a significant production of t h e methylarsenicals. The production of arsenite was evident in several cultures, especially in t h e two coccolithoporids. An extreme situation with respect to the chemical speciation of arsenic was observed in the Salton Sea during a n intense phytoplankton bloom: 90% of the dissolved arsenic was in methylated forms, and only 8% was arsenate, the rest being arsenite. The data presented in this paper show that marine phytoplankton is able to actively take u p arsenate at natural concentrations from its environment, and t o regulate cellular arsenic levels independently of phosphate concentrations over a large concentration range. Significant differences exist between the different algal species studied, both in terms of uptake rates and the type and number of compounds produced. The chemical identity of the u p to 12 organoarsenicals prevalent in the cell extracts remains yet to be discovered. I t has, however, been shown that marine planktonic algae are able to produce the methylarsenic compounds so commonly
Table 111. Arsenic Species in Growth Media and Some Natural Watersa species
diatoms Thalassiosira fluviatilis Skeletonema costatum Cylindrotheca closterium coccolithophorids Cricosphaera carteri Cricosphaera carteri I/ Coccolithus huxleyi dinoflagellates Goniodoma cf. depressurn Gonyaulax polyedra Prasinophyceae(green algae) Platymonas cf. suecica sterile medium surface seawater Salton Sea, surface
dimethyl arslnate
total
0.3 0.2 trace
1.3 1.3 2.4
4.4 1.9 2.4
0.1 13.1 2.2
n.d. 0.5 0.2
n.d. 1.9 1.o
15.5 7.3
13.6 10.4
0.1
0.4
