desorption rate constants using Chironomus tentans larvae (Insecta: Diptera: Chironomidae)

War. Res. Vol. 24, No. 3, pp. 321-327, 1990 Printed in Great Britain.All rights reserved

0043-1354/90$3.00+ 0.00 Copyright © 1990PergamonPress plc

IN SITU DETERMINATION OF PCB CONGENER-SPECIFIC

FIRST ORDER ABSORPTION/DESORPTION RATE CONSTANTS USING CHIRONOMUS TENTANS LARVAE (INSECTA: DIPTERA: CHIRONOMIDAE) M. A. NOVAK 1, A. A. REILLY 2, B. BUSH 2 and L. SHANE2 ~New York State Department of Environmental Conservation, Division of Water, 50 Wolf Rd, Albany, NY 12233 and 2New York State Department of Health, Wadsworth Center for Laboratories and Research, Albany, NY 12201, U.S.A. (First received May 1988; accepted in revised form September 1989)

Abstract--The uptake of polychlorinated biphenyl (PCB) congeners was measured in the larvae of a laboratory-reared chironomid midge, Chironomus tentans, placed in the upper Hudson river, New York during 2 months in 1985. This procedure was investigated as a method for determining water congener concentrations during times of fluctuating PCB levels, and to model uptake of PCBs by river biota. After a 96 h exposure period, total PCB concentrations in the test organisms averaged 6.7 #g g-t total PCBs, compared with water concentrations of 67 ng 1- t (mean value for both months). Uptake and elimination constants, time to equilibrium and concentration factors were calculated for each of 21 selectedcongeners. Analysis of PCB congeners in insects harvested at intervals during the 96 h period showed that uptake differs with varying degrees of chlorination relative to water concentrations. At the end of the exposure period, concentration factors ranged from 4000 to over 300,000 times the water concentrations. Differences in the replicate indicate potential problems with this method as a field tool; instead of using all congeners separated in analysis, several individual congeners should be selected for use based on their importance to the river fauna and the consistency with which they are analyzed. Key words--PCB congeners, kinetic studies, Chironomus tentans, chlorinated xenobiotics, bio-uptake

INTRODUCTION

Several aquatic invertebrates, including freshwater clams (Mollusca: Pelecypoda), chironomid midges (Insecta: Diptera: Chironomidae) and hydropsychid caddistties (Insecta: Trichoptera: Hydropsychida) (Hartley and Johnston, 1983; Bush et al., 1985; Simpson, unpublished results) have been used for biological assessment of aquatic contaminants. These organisms accumulate lipophilic compounds to equilibrium concentrations in excess of those in their environment. Previous investigators have focused on either laboratory determination of uptake kinetics in the presence of constant contaminant concentration or field determination of bioconcentration. Our goal has been to estimate congener (isomer) specific kinetic parameters from time sequence field measurements of both water and organism concentrations. Aside from their intrinsic biological utility, these parameters provide the ability to predict uptake given only waterbody measurements. For most compounds of interest, the relationship between water column concentrations and biological uptake has not been investigated; we anticipate that considerable savings can be realized through in situ determination of these parameters by exposing uncontaminated individuals to contaminated natural environments. 321

The current article reports on a study conducted in the Hudson river, New York, in July and September 1985, using third and fourth instar laboratorycultured Chironomus tentans (Insecta: Diptera: Chironomidae) larvae. The sediments of the upper Hudson river are contaminated with PCBs from capacitor manufacturing operations which discharged for approx. 30 years. These sediments contaminate the water column. The purpose of the study was to document accumulation rates of various PCB congeners in the test organisms. MATERIAI.,S AND METHODS

Field procedures A laboratory culture of Chironomus tentans was maintained, with modifications, according to the methods of Townsend et al. (1981). An attempt was made to eliminate all sources of PCB contamination by using deionized water and glass rearing tanks, and minimizing the use of plastics; Tygon~ tubing was used where necessary. The larval exposures occurred in the upper Hudson river's Thompson Island pool, a reach extending from Fort Edward (at Lock 7) to the dam at Thompson Island. The water was 3 m in depth, and current speed, measured at the exposure site, was 24 cm s -~ in July, and 56 cm s-t in September. Water temperatures were 21.5°C in July and 18°C in September. Larvae were placed in the river in Nitex monofilament nylon screen "envelopes" (mesh opening 560 microns; Tetko, Elmsford, New York), measuring 6.5x 12cm.

M. A. NOVAKet al.

322

Twenty-five third and fourth instar larvae were placed in each cage, and transported to the field in hexane-rinsed galvanized steel pails containing deionized water. The cages were placed, in groups of ten, into steel mesh baskets, which were suspended at a depth of 1 m, from floats anchored by cinder blocks on the river bottom. Triplicate samples were harvested at 0, I, 2, 4, 8, 12, 24, 48, 72 and 96 h. The cages were opened, larvae removed with flexible forceps and counted. The insects were placed in glass vials, the vials capped and put immediately on dry ice. Controls (0 h) were caged and transported to the field, then removed and frozen, without being put into the river. Triplicate water samples were taken at each harvest. Water was collected 0.3 m from the surface, in hexane-rinsed 21. jars fitted with Teflon cap liners. The jars were rinsed three times with river water to equilibrate PCBs on the inner surface, since these compounds adsorb to glass (Bush et al., 1985). Samples were placed on ice for transport back to the laboratory, where they were stored at 4°C until analyzed.

Laboratory methods Larval samples were lyophilized, ground with 2 ml n-hexane in a Tekman Tissuemizer until reduced to a fine powder, then extracted by the method of Bush and Barnard (1982). Water samples were extracted with n-hexane, the extract dried with sodium sulfate and concentrated to 1 ml. All extracts were analyzed on a Hewlett-Packard 5840A gas chromatograph equipped with a 5880 splitless glass capillary inlet, and fitted with a 60m Apiezon L glass column (0.25-0.3 mm i.d.); 76 congeners were separated by this column (Bush et al., 1983). Quantities were calculated using a reference peak compared to external standards of a known congener mixture (200ngml -~ mixture of Aroclors 1221, 1016, 1254 and 1260; 1:1:1:1 in hexane). Recalibration was performed after each set of six samples, by conducting an analysis of the external standard, followed by a hexane sample to detect and remove any impurities left on the column. Congener uptake Twenty-one congeners were selected for study of uptake dynamics during a 96-h exposure in the river. Accumulation of ©ach PCB congener was modeled by utilizing the differential equation governing uptake dynamics: dCt(t) = -- K 2 CL(I ) + K, Cw(t )

(I)

dt

where CL(t)=congener concentration in the larvae (ng g- ~dry wt) at time = t K~ = uptake rate constant (h-~) K2 = elimination rate constant (h -~) Cw(t) =congener concentration in water (ng 1-j) at time = t. Uptake of individual congeners cannot be modeled from equation (1) unless the functional form of Cw(t), the time

specific river concentration, is known. Values for Cw(t) were measured in triplicate at sampling times T~ = 0, T2 = 1. . . . . Tt0 --- 96. Cw(t ) may therefore be approximated with a sequence of linear functions:

Cw(t)=floi+fllit

for

1200 -4-127 1130 +- 429 2440+- 1390" 2870 +- 1250 4020 +- 464 3510+-794 6160+- II00"

*Mean of 2 values.

II0 +- 16 113 +- 9 115+- 13 84 +- 6 lOI +- 23 66+-16 129 +-42

(2)

where fl0~ and fll~ are the estimated intercept and slope parameters for the ith interval. Larval congener concentration at the harvest times, CL(T~), may be modeled by integrating equation (l) utilizing (2): Ki F CL(Ti) = CL(T i ) e-x2r, +_~,lflli(Ti_ T,_te-r2tr,-r,_,))

In equation (3), fl0~and flt~ are determined by Cw(T i_ t) and Cw(T~), so that the only parameters to be estimated, as indicated in equation (1), are K~ and /(2. Water and C. tentans data were subjected to a weighted classical non-linear least squares analysis utilizing equation (3), with weights equal to observed replicate variances. The lack of fit of each datum to the model was examined for statistical significance (studentized residual greater than 2.3), and if found, the observation was deleted. When Cw(t) is relatively constant, integration of equation (1) results in:

Ce(t) = ~KI Cw(/)(1 - e -x2t)•

(4)

After long periods of exposure (t large) the last factor approaches unity so that the expression for larval PCB concentration (CL) becomes Kt/1(2 times the concentration in the river. The time it takes to reach this approximate equilibrium condition depends only on K2, the elimination rate constant. The ratio of K~ to K: is considered the concentration factor of a congener at equilibrium. A practical estimate of equilibrium was defined as being a value within 10% of the true value, as used by Sanders and Chandler (1972). K~ and K2 values, concentration factors and days to equilibrium (within 10%) were estimated for the selected congeners. The number of days required to reach 90% equilibrium for each congener was estimated by setting the last factor in equation (4) to 90%: In 0.10 / 2 4 h days _ K 2 h _ l / d a y (5) RESULTS

P C B composition o f s a m p l e s - - J u l y Total PCB c o n c e n t r a t i o n s in the July water samples ranged from 47 to 166ng1-1 (n = 28 for all 10 exposure times) with a m e a n o f 93 ng 1-~ (Table 1).

Table 1. Total PCB concentrations in Chironomus tentans (ng g- ~dry wt) and water (ngl-~), Hudson river, July and September 1985. Reported values are means of 3 samples +- (rounded to 3 significant figures) 95% confidence limits July September Hour C. tenlans Water C. tentans Water 0 I11 + 124 95+ 19 1110+_69 66+-8 I 815+-677 61 +4 1310_+51 59+- 11 2 584+-508 72+- 11 1660+- 144 37+- 12 4 8 12 24 48 72 96

Tj_I
2120 + 72 2600 _+ 203 3540+- 119 5340 +- 924 6640 +- 553 6480+-1550 7250+- 1840

31 + 40 36 +- 2 36+-9 38 +- 3 27 +- 2 38+-4 33+- I

Determination

of PCB

323

rate constants

T a b l e 2. P e r c e n t c o n t r i b u t i o n o f P C B c o n g e n e r s in r e p r e s e n t a t i v e Chironomus tentans ( a f t e r 8 a n d 4 8 h e x p o s u r e ) a n d w a t e r s a m p l e s ; J u l y a n d S e p t e m b e r 1985, U p p e r H u d s o n f i v e r , N e w Y o r k

C. tentans Water Congener 2 2,2' 26 26,2' 25,2' 26,4' 25,4' 24,4' 24,2'5' 236,4' All o t h e r congeners --Indicates

8h

48 h

July

September

July

September

July

September

-29 16 32

-10

13 10 . 6

---

5 --

--

--

------

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5 6 7 7

5 -6 6

20

50

67

75

78

3

.

. 15 . . -5 5 --

. .

65

. . .

. .

congener was not 1 of 4 most abundant for that sample.

The water samples were characterized by the abundance of three congeners: 2,2'-dichiorobipbenyl (2,2'), 26-dichlorobiphenyl (26) and 26,2'-trichlorobiphenyl (26,2') (Table 2). These three together contributed from 51 to 78% of the total PCB concentration. The next most abundant congener was 25,2'-trichlorobiphenyi (25,2') (Table 2). The congener pattern of PCBs in C. tentans differed substantially from that in the water (Fig. 1; Table 2). Total PCB concentrations for the insects ranged from 3 3 n g g -~drywt in the control samples (mean of 111 ng g- ~ for three replicates) to 7540 ng g- ] after 96 h (mean of 6160 ng g-~ for three replicates). The water pattern was dominated by 2 or 3 di- or trichlorinated congeners, contributing approx. 50% of the total concentration. The C. tentans samples

were characterized by a greater number of congeners with the most abundant contributing only 5-10% (Table 2). From 1 to 24 h, 2,2' and 26,2' were often most abundant congeners, as in the water samples. However, at 48, 72 and 96h, neither of these compounds was dominant in the larvae. Congeners taken up more slowly, such as 24,2'5'-tetrachlorobiphenyl (24,2'5'), 236,4'-tetrachlorobiphenyl (236,4') and other tri- and tetrachlorinated congeners, were most abundant in 14 of 18 samples, although each contributed less than 10% of total PCB concentration. Congener uptake--July

For each of the congeners of interest, and for total PCBs, K~ and K2, concentration factors, and days to HUDSON RIVERWATER (Sample 85-77, July exposure) 104 ng/I

(A)

(s)

~ i o

I 22.~

Chironomu: tentans

urs)

I 5o.24

I 7e.ls

] ~o~.ss

ELUTION TIME (mln)

Fig. 1. Sample ehromatograms from Hudson river water (A) and Chironomus tentans (B) PCB analyses, showing patterns of congener abundance.

324

M.A. NOVAKet al.

equilibrium are shown in Table 3. The congener specific uptake and estimated exponential model are illustrated in Fig. 2. K~ values were large and positive; K2 values were less than one in nearly all cases, indicating that the ability of the test organisms to eliminate most congeners was limited. K 2 values decreased with increasing chlorination, consistent with the more lipophilic nature of the highly chlorinated congeners. Concentration factors (CF) ranged from approx. 4000 to 300,000 (Table 3). September results

Reported 0 h total PCB concentrations in the test organisms (mean 11 l0 ng g-J) were approximately an order of magnitude higher than the mean in July; 2-chlorobiphenyl and 2,2'-dichlorobiphenyl were particularly elevated. The source of this difference is unknown, but contamination in the laboratory colony itself was eliminated by analyzing insects and water taken directly from the rearing tanks. Samples of food and organic debris collected from the tanks were also analyzed, but elevated PCB levels were not detected. Several congeners: 24,2'; 234,4'; and 245,2'5', which separated satisfactorily in the July analysis, were not present in the chromatograms from the September exposure. Also, as seen in Table 1, the September total PCB water concentration was nearly constant over time at approx. 35 n g g - J , less than every July value. DISCUSSION In the July exposure, the range of Kj and K 2 values calculated for the selected congeners is consistent with the values calculated by Muir et al. (1983) from a laboratory study of 245,2'4'5'-hexachlorobiphenyl

uptake by Chironomus tentans. They reported that for this congener, equilibrium was not reached after 96 h. Similarly, some of the more highly chlorinated congeners in the present study did not attain equilibrium during the 96 h exposure period. With additional method development, K 1 and K2 uptake and elimination constants may be useful in predicting PCB levels expected in a test organism, such as C. tentans, from water column concentrations, and provide a model for predicting concentration patterns in other river organisms. In this study, the July replicate was successful in defining the C. tentans/water PCB relationship, but the September replicate was unsuccessful. Detailed analysis of the September exposure revealed that, unlike the July results, two or even all three of the values do not agree with equation (3), and an assumed normal distribution for the residuals. Calculated K 2 values for four congeners: 236,3'; 235,2',6'; 25,3'4'; and 24,3'4', were negative. These negative values result from an inability to identify sets of outlying points; the data are not sufficiently self-consistent to identify them from outliers. Between the two replicates, no congener was found to have Kt and K 2 statistically indistinguishable from July to September. These are noted in Table 3. Statistical tests were conducted (P <0.05) to compare the estimated parameters between replicates. For seven congeners, the Kj estimates were not significantly different and for six other congeners, the K 2 values were not significantly different. Since contamination of the laboratory colony was ruled out as a reason for the unexpected September results, analytical error was suspected as the cause of the discrepancies. The September results were included here to point up potential problems with this method of congener uptake measurements.

Table 3. Uptake and elimination constants, concentration factors (ng g- ~dry wt), and estimated time to reach equilibrium (rounded to 3 significant figures) for Chironomus tentans larvae in the Thompson Island pool of the Hudson river, July 1985 PCB KI CF K2 Days to 90% congener ( u p t a k e / h ) ( K I / 1 ( 2 ) (elimination/h) equilibrium 2,2' 2370 5830 0.406 0.2 26* 1040 4190 0.247 0.4 26,2'* 6300 5950 1.060 0.1 2,4't 10,600 116,000 0.091 1.1 25,2'* 1860 37,000 0.050 t .9 24,2' 1930 56,700 0.034 2.8 26,4't I 120 47,800 0.023 4.1 246,2't 1730 42,800 0.041 2.4 25,3't 1920 156,000 0.012 7.8 25,4'¢ 4390 258,000 0.017 5.6 24,4'* 5670 175,000 0.032 3.0 25,2'5' 3590 77,500 0.046 2.1 24,2'5'* 4670 117,000 0.040 2.4 24,2'4'* 4840 131,000 0.037 2.6 236,3' 2500 208,000 0.012 8.0 236,4't 5610 138,000 0.041 2.4 235,2'6' 5080 177,000 0.029 3.3 25,3'4' 8120 294,000 0.028 3.5 24,3'4'* 6660 362,000 0.018 5.2 234,4' 18,500 336,000 0.055 1.7 245,2'5' 2550 141,000 0.018 5.3 *, tCongeners for which Kt (*) or/(2 (t) values calculated for the September exposure were not significantlydifferent from values calculated for July results.

325 Determination of PCB rate constants

2 4.'

2.5

0.5

~,a"~O

0

20

22

24

26

28

30

32

34.

36

38

4.0

.,"o

"

,t~',3~

x',-

RETENTIONTIME IN MINUTES

2.5

. ~.3~,Or'

,=2°~

•

o.s o

" "l, 4.8

4.8

5o

•

•

=" ~2

,~

~6

~ 4 ° s8

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RETENTIONTIME IN MINUTES

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.,

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.,/,

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RETENTION TIME IN MINUTES

Fig. 2. July congener specific uptake and estimated exponential model.

326

M.A. NOVAKet al.

The validity of the method depends on how closely organism concentrations can be predicted from water concentrations by use of equation (3). Muir et al. (1983) found that first order kinetic rate equations as used here were appropriate for describing organism uptake. The exact solution to differential equation (1):

CL( T) = KI .fCw(z) e -x2(r-~) dz has been approximated by assuming Cw(t) is linear between sampling times; the sampling intervals must be sufficiently close together for the approximation to be accurate. The integral in the above equation points out a possible advantage in examining the organism concentrations: they are the total response to the input function Cw(t). The water concentration may be highly irregular at times when PCB load is rapidly changing, as during continued disposal, natural sediment scouring or during river dredging. Under these conditions there is no assurance that sequential water samples will capture transient concentration peaks and troughs. Previous studies (Derr and Zabik, 1974; Lynch and Johnson, 1982; Macek et al., 1979) have shown that for several other organic compounds, uptake in invertebrates is almost entirely from the water column rather than from dietary sources. Comparison of CL and Cw may therefore provide verification that no significant peaks are being missed. Once uptake constants have been fully defined, use of Chironomus tentans to delineate water concentrations of biologically important congeners during the period of exposure will be another appropriate use for this method. Since these congeners are preferentially accumulated by organisms, resulting in high concentrations relative to water, the problems of direct measurement of low or fluctuating water column concentrations would be eliminated. This is illustrated by comparison of results of the present study with those of previous studies. The PCB concentrations in the water samples differ from those reported by Bush et al. 0985) for water samples collected in the Thompson Island pool in 1983. They reported substantially higher total concentrations (approx. 500 ng l-1), and found 2-chlorobipbenyl (2) to be one of the most abundant congeners at this location. In the present study, 2-chlorobiphenyl was not present above the limits of detection in any of the water samples, although it was detectable in some C. tentans samples. Wood et al. (1987) expressed caution about the use of aquatic organisms for monitoring xenobiotic compounds such as PCBs, since congener uptake varied both among and within species. However, within one model species, differences in congener uptake are problematic only if total PCB concentrations are measured, but are not a limitation if congener-specific analysis is conducted. In selecting specific congeners for use in predicting PCB uptake in river organisms, congeners which yielded consistent results should be

used, in the absence of further testing. Several of the congeners studied--24,2'; 23,2'; 23,2'3'; 234,4'; and 245,2'5'--were not consistently separated by the Apiezon L column. Since the less lipophilic congeners 2,2'; 26; 26,2'; and 2,4' are more water soluble and appear to be variable in the environment, they are considered less desirable for use in this manner. However, four congeners---26,4'; 246,2'; 25,4'; 24,4'; and 236,4'--had comparable Ks values in the July and September replicates, and the differences in K] values, while statistically significant, were small enough to result in the estimated concentration factors being within a factor of 4 (246,2') or less. These congeners should be considered for further predictive studies. This method has potential usefulness in field applications requiring biological assessment tools. If some contaminants are known to be preferentially concentrated, it provides a means of elucidating concentrations to be expected in the natural biota from either hypothesized or observed inputs [Cw(t)]. Placing test organisms both within and outside a contaminated area could be used to delineate concentrations found in specific river organisms. Additionally, this method could be used to measure initial concentrations in organisms, and those obtained after clean-up procedures are conducted. Acknowledgements--This study was supported by a grant

from the United States Environmental Protection Agency to the New York State Department of Environmental Conservation's Hudson River PCB Reclamation Demonstration Project. The research was initiated by Dr Karl Simpson, of the New York State Department of Health, now deceased. We thank Lawrence Abele for field collection and processing of the samples, and Robert Bode for assistance throughout the project. REFERENCES

Bush B. and Barnard E. (1982) Determination of nonpolar chlorinated hydrocarbons and PCB in microsamples. Analyt. Lett. 15(A20), 1643-1648. Bush B., Connor S. and Snow J. (1983) High resolution gas chromatographic analysis of nonpolar chlorinated hydrocarbons in human milk. J. Ass. off. analyt. Chem. 66, 248-255. Bush B., Simpson K. W., Shane L. and Koblintz R. (1985) PCB congener analysis of water and eaddisfly larvae (Insecta: Trichoptera) in the Upper Hudson River by glass capillary chromatography. Bull envir, contain. Toxic. 34, 96-105. Derr S. K. and Zabik M. J. (1974) Bioactive compounds in the aquatic environment: studies on the mode of uptake of DDE by the aquatic midge, Chironomus tentans (Diptera: Chironomidae). Arch. envir, contain. Toxic. 2, 152-164. Hartley D. M. and Johnston J. B. (1983) Use of the freshwater clam, Corbicula manilensis as a monitor for organoehlorine pesticides. Bull. envir, contain. Toxic. 31, 33--40.

Lynch T. R. and Johnson H. E. (1982) Availability of a bexachlorobipbenyl isomer to benthic amphipods from experimentally contaminated natural sediments. In Aquatic Toxicology and Hazard Assessment (Edited by Pearson J. G., Foster R. B. and Bishop W. E.), pp. 273-287. American Society for Testing and Materials, Philadelphia, Pa.

Determination of PCB rate constants Macek K. J., Petrocelli S. R. and Sleight B. H. III (1979) Considerations in assessing the potential for, and significance of, biomagnification of chemical residues in aquatic food chains. In Aquatic Toxicology (Edited by Marking L. L. and Kimerle R. A.), pp. 251-268. American Society for Testing and Materials, Philadelphia, Pa. Muir D. C. G., Townsend B. E. and Lockhart W. L. (1983) Bioavailability of six organic chemicals to Chironomus tentans larvae in sediment and water. Envir. Toxic. Chem. 2, 269-281. Muir D. C. G., Townsend B. E. and Webster G. R. B. (1985) Bioavailability of L4C-1,3,6,8-tetrachlorodibenzo-p-dioxin and ~4C-octachlorodibenzo-p-dioxin to aquatic insects in sediment and water. In Chlorinated Dioxins and Dibenzt~furans in the Total Environment H (Edited by

W.R. 24/3---E

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Kerth L. H., Rappe C. and Choudhary G.). Butterworth, Boston, Mass. Sanders H. O. and Chandler J. H. (1972) Biological magnification of a polychlorinated biphenyl (Aroclor 1254) from water by aquatic invertebrates. Bull. envir, contain. Toxic. 7, 257-263. Townsend B. E., Lawrence S. G. and Flannagan J. F. (1981) Chironomus tentans Fabricius. In Manual for the Culture of Selected Freshwater Invertebrates (Edited by Lawrence S. G.). Canadian Special Publication of Fisheries and Aquatic Sciences No, 54. Department of Fisheries and Oceans, Ottawa. Wood L. W., Rhee G-Y., Bush B. and Barnard E. (1987) Sediment desorption of PCB congeners and their biouptake by dipteran larvae. Wat. Res. 21, 875-884.