Accumulation of heavy metals by some solitary tunicates

GORDON P. DANSKIN Department yfBiologica1 Sciences, Simon Fraser University, Burnabg, B.C. , Canada V5A IS6

Received June 24, 1977

DANSKIN, G. P. 1978. Accumulation ofheavy metals by some solitary tunicates. Can. J . Zool. 56: 547-55 1.

The vanadium contents of eight species of solitary ascidians for which quantitative data are lacking are reported. It is observed that the occurrence of the metal within the class does not conform to taxonomic groups, but instead occurs sporadically throughout. For example, the phlebobranch Chelyosoma productum was observed to contain 800 ppm vanadium, whereas in a second member of the same family. Corella willmeriana. the metal could not be detected. Corella willmerianu contained a substantial iron concentration. The stolidobranch Boltenia villosa was found to contain vanadium at a level previously observed only among the phlebobranchs.

The vanadium content of Ciona intestinalis shows a linear dependence upon the dry weight of the animal, and can be estimated from a concentration value of - YO pg vanadium per gram dry weight, independent of the size class of the animal. No significant seasonal variation in the vanadium concentration of C . intestinalis was observed.

DANSKIN, G. P. 1978. Accumulation of heavy metals by some solitary tunicates. Can. J. Zool. 56: 547-55 1.

On trouvera ici des donnees quantitatives inedites sur le contenu en vanadium de huit especes d'ascidies solitaires. La presence du metal n'est pas reliee au groupe taxonomique, mais semble plutBt sporadique. Par exemple, le phlebobranche Chelyosoma productum contient 800ppm de vanadium, alors que Corella willmeriana, pourtant de lam&me famille, n'en contient pas du tout. Chez C. willmeriana, il y a une forte concentration de fer. Les specimens du stolidobranche Boltenia villosa examines contiennent du vanadium en concentrations rencontrees d'ordinaire seulement chez des phlebobranches.

Le contenu en vanadium de Ciona intestinalis est en relation directe avec le poids sec de I'animal et peut Etre estime a partir d'une concentrat~on d'environ 90pgIg poids sec, indepen- darnment de la classe de taille de I'animal. I1 ne semble pas y avoir de variation saisonniere de la concentration de vanadium chez cette espece.

[Traduit par le journal]

Introduction information regarding the vanadium levels accumu- Among the biochemical oddities which distin- lated under normal conditions by the various size

guish the ascidians from all other animals is their classes of agiven species. Therefore, the vanadium ability to concentrate vanadium in blood cells at content of the much studied ascidian, Ciona intes-

levels 100000 to ,000000 times that of seawater tinalis. was determined as a function of its dry

(Rumme] et a[. 1966). webb (1939) suggested that weight in order to provide base-line information for

this facility the ancestral tunicate, the culturing of ascidians in vanadium-free media. but is retained only in those families constituting Because Bertrand (1950) and Levine (1962) had the primitive order phlebobranchia. hi^ study was postulated seasonality in the vanadium contents of

undertaken to ascertain whether solitary tunicates ascidians, evidence was sought by sampling the which contain vanadium conform to phy]etic Same species, C. intestinalis, at different times of

groups. the year. Remarkably little is known about the physiologi- Materials and Methods

cal significance of the vanadium blood pigment, Solitary tunicates representing four Pamilies were collected although it has been shown to be a strong reducing intertidally and subtidally at Bamfield, British Columbia (48"501

agent (Koval'skii and Rezayeva 1964; Rezayeva N , 125'8' W) during the spring and summer of 1976. These included Ascidiu ceratodes (Huntsman) and A. parafropa 1964; Bie'ig et 1966)' A IllJmber of studies (Huntsman) from the order Phlebobranchia, family Ascidiidae;

vide evidence that the are Chelyosoma producrum Stimpson and Corella willmeriana involved in test formation (Endean 1955; Smith Herdman, order Phlebobranchia, family Corellidae; Boltenia 1970; Goodbod y 1974). However, to date no r~illosa (St impson), Halocynthia aurantium (Pallas), H . igaboja

studies have been performed in ascidians are Oka, and Pyura haustor (Stimpson) from the order Stolidobran- chia. family Pyuridae; and Cnemidocarpajnmarkiensis (Kiaer)

reared in vanadium-free Or vanadium-depleted and Styela montcreyensis (Dall), order Stolidobranchia, family seawater to assess the requirement for the metal. Styelidae. Only solitary tunicates were used for metal analysis This situation can in part be attributed to the lack of because the test, frequently encrusted with epizoites and sedi-

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548 CAN. J. ZOOL.

nient, is easily removed. Cionri intestintrlis (Linnaeus) was col- lected subtidally at Venice, California (34"59' N, 118"28' W) in winter and summer 1976. This particular species was examined for its vanadium content asa function of weight class and season of collection since i t is the only species for which an average vanadium concentration based on a large sample size is available (Bielig et (11. 1961h).

Animals used for metal assays were squeezed gently to expel extraneous seawater from the branchial chamber. Each speci- men was dissected from its test over a weighed scintillation vial to prevent loss of blood and fluids. The stomach and intestine were opened to remove gut contents which frequently included sediment. After drying to constant weight at 1 IWC, specimens were digested in a 2:l solution of hot concentrated HNO,- H2S0,.

Vtrnac(irrtn Detrrtninations Colorimetric analysis for vanadium was performed using the

phosphotungstate method (Wright and Mellon 1937) with NH,VO, as standard. The method of standal-d additions (Chris- lian and Feldman 1970) was used in case the presence of organic and inorganic residues from solubilized ascidian tissues either suppress or enhance the development of color in experimental preparations. Absorbance at 430 nm was measured on a Zeiss PMQ I1 spectrophotometer.

For gravimetric determination of vanadium as V,O, the metal was extracted as the 8-quinolinol derivative in order to eliminate iron. copper, and other possible interfering ions (Talvitie 1953). The organovanadium compound was fused and oxidized to con- stant weight at 800°C. Percentage recovery and effectiveness of the separation were assessed with appropriate aqueous stan- dal-ds.

Iron Drtertninrrtions Colorimetric determinations of iron were made using the

rhiocyanate method (Sandell 1959) with FeCI,.6H20 as internal standard (Christian and Feldman 1970).

Results The vanadium concentrations of ten species of

solitary ascidians are reported (Table I ) , and among these are eight for which previous quantita- tive data are lacking. Vanadium was found in As- cidia ceratodes, A. paratropu, Boltenia villosa, Chelyosoma productum, Halocynthia igahoju, and Styela montereyensis but was not detected in Cnemidocurpa$nmarkiensis, Corella willmeriana, Hulocynthia urrranthm, or Pyura huustor. Bol- tenia villosa, determined gravimetrically, con- tained 520 ppm dry weight vanadium (Table 2) .

The addition of vanadate standard to prepara- tions of Ciona intestinalis and Corella willmeriana yielded Beer Lambert curves having the same slope as aqueous vanadate (Fig. 1).

There is a positive correlation between the dry weight of individual specimens of Ciona intes- tinalis and the vanadium content (Table 3). The metal content shows a linear dependence upon dry weight, and regression coefficients which have the units of concentration (kg vanadium per gram dry weight) were calculated for two samples individu- ally (Fig. 2). Differences between the regression coefficients are not significant and a regression coefficient was calculated for the pooled data

TABLE 1. Vanadium concentration of solitary ascidians (ppm dry weight)

Number of animals Vanadium

S~ecimen ~ o o l e d concentration

Order Phlebobranchia Family Ascidiidae

Ascidia parafropa 1 3750' 850b Ascidia ceratodes 2 1700' 2250b Ascidia ceratodes 1 4500 1300'

Family Corellidae Corella willmerianu 2 d d d d - - - - Corella willmeriana 10 - d

Chelyoson?a producturn 8 800

Order Stolidobranchia Family Pyuridae

Pyura l~austor 1 d - Boltenia villosa 5 750 Halocynfhia igaboja 1 175 Halocynthia airrantirrrn 1 - ,I

Family Styelidae Slyela montereyensis 2 40" 36' Of Cnemidocarpafinmarkiensis 1 - d

.Blood and body fluids only. 'Tissues with fluids drained. <See Swinehart et a / . (1974). Based on weight o f whole animal, including

test. dNot detectable ( < 1 pg vanadium). eTwo different samples, each of two animals pooled. ,See Goldbera el a/. (1951). Based on weight of whole animal including

test. Vanadium determined with 8-quinolinol (oxine).

TABLE 2. Vanadium concentration of Boltenia villosa (ppm dry weight)

Vanadium Vanadium Sample recovery concentration

25 pg V, 500 pg Fe 23 ~g (897,) 500 pg Fe - Bollenia cillosa" 13 pgb 520'

750d

aDrv weieht 0.025 a. b~s;umi"-g~89~ y i z d .Determined g;avimetrically. dDetermined spectrophotometrically (Table 1) using the same sample o f

0.025 g dry weight (5 animals pooled).

(Table 4 and Fig. 2). Since the dependence of metal content upon dry weight is linear, the vanadium concentration is a constant, independent of animal size. As a second estimate of vanadium concentra- tion, mean values were collected from raw data for each sample and were compared with F-test (Table 5) . Difference between the means is not significant, and a mean concentration was calculated for pooled data (Table 5) .

Based on these two estimates of the vanadium concentration (Tables 4 and 5 ) , the vanadium con- tent of an individual specimen is given by:

[ 11 Vc /,- 90 Wc

where Vc is the vanadium content of Ciona intes- tinalis in micrograms, and W c is the dry weight of- the specimen in grams.

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DANSKIN

DRY WEIGHT (9)

VANADIUM ADDED (pg)

FIG. 1. Absorbance of phosphotungstovanadate. Am- monium vanadate as aqueous standard; 0 standard additions to Ciona intestinalis; standard additions to Corella willmeriana. Deviations of the three slopes are not significant (p > 0.01).

TABLE 3. Correlation of dry weight of Ciona intestinalis to vanadium content

Sample Date of Correlation

collection coefficient ( r )

Jan. 1976 0.94"(n = 32) June 1976 0.84" (n = 32)

probability << 0.005.

that the sample derived from population where

The mean vanadium concentrations of winter and summer samples of Ciona intestinalis, col- lected from the same location, are not significantly different at the 0.95 confidence level (Table 5).

Corella willmeriana, which contains no detect- able vanadium (Table l), was found to contain 430 ppm iron (Fig. 3).

Discussion Webb (1939) concluded that the assemblage of

families in which vanadium is found corresponds to the order Phlebobranchia, and he predicted that phlebobranchs for which no data were available would contain vanadium as well. This study reports for the first time a member of the order Phlebobran- chia, Corella willmeriana, which contains no de- tectable vanadium. Prior to this report, the lowest vanadium concentration found in a phlebobranch was 100- 160 ppm in Ciona intestinalis (Goldberg et al. 1951 ; Bielig et al. 1961b).

FIG. 2. Vanadium content of Ciona intestinalis: collected Jan. 1976; A collected June 1976. - - Regression of January data; slope (b ' , ) = 102.1 pg Vlg dry weight, intercept (a',) = - 1.5 pg V. -Regression of June data; slope (b',) = 83.4 pg Vlg dry weight, intercept (a' ,) = 1.7 pg V. The slopes are not significantly different at the 0.95 confidence level. - Regres- sion of pooled data; slope = 90.3 pg Vlgdry weight, intercept = 0.3 pg V. Each point represents the average of determinations made in duplicate.

TABLE 4. Vanadium content of Ciona intestinalis determined by linear regression analysis

Regression coefficient (b',) Intercept (a'.)

Sample (1.18 V/g dry weight) V)

Jan. 1976 102.1" -1 .5 June 1976 83.4" 1 . 7 Pooled 90.3 0 . 3

.The regression coefficient (b'd falls within the 0.95 confidence level of (b.2).

TABLE 5. Mean vanadium concentration of Ciona intestinalis (ppm dry weight)

Sample Mean .-

Jan. 1976 82.1" (n = 32) June 1976 101.9" (n = 32) Pooled 92.0 (n = 64)

.The difference between the means is not significant at the 0.95 level; F,(0.00718) << F,.,,[l, 301.

According to Webb (1939), the ancestral form which gave rise to the present day orders Stolido- branchia and Aplousobranchia lost the ability to accumulate vanadium. Nevertheless, in this inves- tigation the stolidobranch Boltenia villosa was found to contain 520-750 ppm vanadium. Compar- able high levels of vanadium are normally restricted

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550 CAN. J . ZOOL. VOL. 56, 1978

IRON ADDED (pg)

FIG. 3. Iron content of Curella willmeriunu determined with standard additions of ferric chloride. Sample (1.79 mg) contains 0.77 pg Fe = 430 ppm.

to two families of the order Phlebobranchia: the Ascidiidae, and the Perophoridae (Goodbody 1974). In the past, relatively low vanadium con- centrations have been reported for the stolido- branchs Distomus varialosus (Gartner) (131 ppm) (Bertrand 1950) and Molgula manhattensis (De- Kay) (100 ppm) (Carlisle 1958). Subsequently the analytical techniques of Bertrand (1942) have been questioned (Talvitie 1953), while recent studies of M. manhattensis reported <20 ppm vanadium (Swinehart et ul. 1974). Boltenia villosu therefore appears singular as a stolidobranch which contains a substantial vanadium concentration.

Webb (1939) concluded that vanadium accumu- lation was restricted to the order Phlebobranchia and considered it very unlikely that cells as specialized as vanadocytes would have arisen more than once within the group. However, it has been shown that vanadium accumulation occurs in some aplousobranchs (Goldberget al. 1951; Levine 1962; Ciereszko et al. 1963; Swinehart et al. 1974) and consequently Millar (1966), in his modern evolu- tionary treatment of the Ascidians, affords a posi- tion of systematic isolation to the genus Euherdmania partially on the strength of its ability to concentrate vanadium. The adaptation to diffe- rent metals or to different oxidation states of van- adium (Endean 1955; Kokubu and Hidaka 1965; Smith 1970; Swinehart et al. 1974; Kustin et al. 1976) has led Swinehart et al. (1974) to hypothesize that vanadium concentrations, and more specifi- cally vanadium oxidation states, follow taxonomic patterns.

The observation that the phlebobranch Corella willmeriana contains no detectable vanadium while

the stolidobranch Boltenia villosa does, con- tradicts the putative theory proposed by Swinehart et al. (1974). Rather than being restricted to taxonomic groupings, it would appear that the abil- ity to accumulate vanadium has arisen (or alter- nately has been lost) a number of times within the class Ascidiacea, and that within families there is no consistency in the distribution of vanadium (Table 1). Levine (1962) has suggested that the switch from one metal to another may be associated with relatively small genetic, physiological, or ecological change.

Prior to this report, no one has explored the possibility of a relationship between vanadium con- tent and size of ascidian accumulating this metal. In this investigation it was found that the vanadium content of Ciona intestinalis shows a linear depen- dence upon the dry weight of the animal (Fig. 2), and an estimate of its content is given by Eq. 1 above. This value of 90 pg vanadium per gram dry weight is in agreement with previous studies of pooled specimens which were found to contain 100-160 ppm vanadium (Goldberg et al. 1951; Rummel et al. 1966). With this information in hand, it is now possible to ascertain whether ascidians maintained in vanadium-depleted seawater can still accumulate the metal to normal levels, and if not, whether they show any type of deficiency symp- toms. Vanadium is a necessary growth factor in rats (Schwarz and Milne 1971), yet this same metal, concentrated to such high levels and postulated to have so many biological activities, has never been proven to be essential in the ascidians.

In the past it has been noted that smaller weight classes of ascidian take up radiovanadium faster than do larger classes (Bielig et a / . 1961~) . At first, this observation might suggest that smaller animals should contain proportionately more vanadium than larger ones, resulting in a nonlinear relation- ship between metal content and dry weight. How- ever, it is known that the ascidians 'turn over' rather than store all vanadium which is taken up (Kustin et al. 1975). Therefore, the relationship observed in this study would imply that smaller animals have a faster turnover rate than do larger ones in order to preserve the linearity observed. Faster turnover in smaller size classes might result from a lower ratio of the branchial chamber volume to its adsorptive surface.

Bertrand (1950) has speculated that vanadium is preconcentrated by the plankton upon which the ascidians feed and that seasonal differences in the composition of plankton and seawater may result in changes in the vanadium levels of the tunicates as well. However, when Ciona intestinalis was sam-

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DANSKIN 551

pled in the same location in summer and in winter there was no significant difference in metal con- centration. This lack of seasonality is not surprising since its uptake is regulated by the organism, rather than by its availability in the medium (Kustin et al. 1975; McLeod et al. 1975). Not until the supply of vanadium fell so far below its normal level of 3 ppb in the sea (Burton 1966; McLeod et al. 1975) that availability rather than efficiency of assimilation became limiting, could seasonal variation in van- adium resources affect the concentration observed in the ascidians.

Summary 1. The previously held notion that all phlebo- branchs concentrate vanadium while stolido- branchs have lost the facility is incorrect. 2. There is a linear relationship between vanadium content of Ciona intestinalis and the dry weight of the individual specimen. This observation provides a base line for studies of the animal reared in vanadium-free or vanadium-depleted artificial sea- water. 3. No evidence was found for seasonal differences in vanadium concentrations which is in accord with previous observations that uptake is regulated by the animal and is not affected by the availability of vanadium above normal levels.

Acknowledgments I wish to thank my supervisor, Dr. P. V.

Fankboner for his assistance and encouragement throughout this study. In addition, I am grateful to Dr. M. J. Smith for helpful discussions and for critical reading of this manuscript. This investiga- tion was supported by National Research Council of Canada grant A6966 to Dr. P. V. Fankboner, Simon Fraser University.

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