Interlaboratory calibration results from chlorinated hydrocarbon analyses in marine sediments

155

Marine Chemistry, 4(1976) 155--163 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

INTERLABORATORY CALIBRATION RESULTS FROM CHLORINATED HYDROCARBON ANALYSES IN MARINE SEDIMENTS*

S. P. PAVLOU and W. HOM

University of Washington, Department of Oceanography, Seattle, Wash. (U.S.A.) (Received August 12, 1975; revision accepted November 24, 1975)

ABSTRACT Pavlou, S. P. and Horn, W., 1976. Interlaboratory calibration results from chlorinated hydrocarbon analyses in marine sediments. Mar. Chem., 4: 155--163. The reliability of chlorinated hydrocarbon measurements in natural marine sediments is assessed statistically for the various sample treatments and quantitation techniques used by ten laboratories. For polychlorinated biphenyls, a standard deviation within -+ 25% of the mean can be expected. Methodology and spectral analysis are the major factors responsible for the variability. Extraction from dry sediment gives the greatest recovery, and measurement of chlorobiphenyl concentrations by homologs alleviates the effect of internal component changes. INTRODUCTION

Although investigations of the transport and bioaccumulation of chlorinated hydrocarbons (CH) in the marine environment are continuing, the reliability of analytical methodology has not been rigorously evaluated. In two earlier publications describing the results of separate intercalibration experiments (Holden, 1973; Harvey et al., 1974), sample treatment and analysis techniques which are critical for assessing data reliability have not been adequately discussed. We have completed a more detailed investigation to isolate the factors responsible for the commonly observed variability in CH analysis. In order to achieve statistical significance we have included ten laboratories (Table I). This paper evaluates the results statistically and compares the methodology. METHODS

The sediment samples were obtained from EUiott Bay, Washington, with a 0.1-m 2 galvanized steelvan Veen grab sampler and were immediately frozen. *Contribution No. 886 of the Department of Oceanography, Universityof Washington.

156 TABLE I List of participants D. J. Baumgartner, A. Yartzoff and M. Feldman J. N. Blazevich

C. S. Giam, H. S. Chan and J. P. Kakareka G. R. Harvey and H. Miklas C. E. Olney and T. F. Biddleman S. P. Pavlou and W. Horn J. Phillips, A. Edwards and P. Murphy R. W. Risebrough and B. DeLappe V. Stout, L. Lewis and C. Houle D. R. Young and T. Heeson

U.S. Environmental Protection Agency Coastal Pollution Branch, National Research Center, 200 S.W. 35th St., Corvallis, Oreg. 97330, U.S.A. U.S. Environmental Protection Agency Region X Laboratories, U.S. Army Terminal, 1555 Alaskan Way S., Seattle, Wash. 98134, U.S.A. Department of Chemistry, Texas A & M University, College Station, Texas 77843, U.S.A. Woods Hole Oceanographic Institution, Woods Hole, Mass. 02543, U.S.A. Department of F o o d and Resource Chemistry, University of Rhode Island, R.I. 02881, U.S.A. Department of Oceanography, University of Washington, Seattle, Wash. 98195, U.S.A. Hopkins Marine Station Lab., Stanford University, Menlo Park, Calif. 94305, U.S.A. Bodega Marine Laboratory, University of California, Box 247, Bodega Bay, Calif. 94923, U.S.A. National Marine Fisheries, 2725 Montlake Blvd. East, Seattle, Wash. 98195, U.S.A. Southern California Coastal Water Research Project, 1500 Imperial Highway, E1 Segundo, Calif. 90245, U.S.A.

The samples were then thawed and placed into a clean 3-1 Pyrex beaker and thoroughly homogenized with a motor-driven stainless-steel stirrer. Aliquots of homogenized sediment were immediately placed into a clean 16-oz. jar, frozen and sent to the participants for analysis. Each laboratory was asked to report the CH concentrations, the methods used, and to submit representative chromatograms of the sainple and standards. A detailed documentation of this information was presented elsewhere (Hom and Pavlou, 1975).

R E S U L T S A N D DISCUSSION

The concentrations for total chlorobiphenyls, (2; PCB), total pesticides, (Z DDT), and other c o m p o u n d s are summarized in Table II. The standard deviation for ~ D D T was as large as the mean. This is n o t unusual since Z DDT in the sample comprised only 3% of the Z PCB content and interference b y PCB's hindered their identification and measurement.

3 4 2

A B C

2

1 1 1 1

F .4

G H I J M e a n o f all quantitations

1 1 2 1 22

2

1 1

1 1 2

Number of q u a n t i t a t i o ns/ extraction

207 529 683 633 486 532 695 720 612 +- 134

-+ 0

-+ 29 ± 49

6 1 3 _* 21 551 +_ 33 765 +_ 46

r.PCB

64 68 NA NA 2 0 . 3 2 +_ 1 7 . 9 4

6 . 2 3 -+ 1.02

NA

5 18.75 2 7 . 5 0 + 4.53

2: D D T .3

NA NA NA NA

NA

NA NA Dieldrin 0 . 8 6 -+ 0.09 Cis-Chlordane 2 . 3 8 +_ 0 . 5 9 T r a n s - C h l o r d a n e 2.21 -+ 0 . 6 2 NA

other compounds

Mean CH c o n c e n t r a t i o n s ( 1 0 -9 g/g d r y w e i g h t ) .2

,1 T h e l a b o r a t o r y d e s i g n a t i o n s were r a n d o m l y assigned. ,2 T h e e r r o r given is t h e s t a n d a r d d e v i a t i o n b a s e d o n t h e n u m b e r o f analyses r e p o r t e d . ,3 S u m o f D D T m e t a b o l i t e s r e p o r t e d . ,4 T w o m e t h o d s o f q u a n t i t a t i o n r e p o r t e d , I a n d H, respectively. See t e x t . NA, N o t available.

1 1

D E

:

Number of extractions

Laboratory*

Summary of data

T A B L E II

158

A statistical treatment of the data shows that the reliability in this study is consistent with the observations of Holden (1973) and Harvey (1974). The ratio of the standard deviation to the mean, o/~, for PCB's in the three investigations are compared in Table III. The agreement is good, suggesting that T A B L E III C o m p a r i s o n o f d a t a variability a m o n g several i n t e r l a b o r a t o r y c a l i b r a t i o n studies Investigator

Participants

a/~

Holden (1973) Harvey e t al. ( 1 9 7 4 ) This s t u d y

26 6 10

0.25 0.21 0.22

T A B L E IV S u m m a r y o f results f r o m o n e - w a y analysis of v a r i a n c e S u m o f squares

D F .1

M e a n square

F .2

Between laboratories Within laboratories

363936 13911

10 11

36393 1265

28.8

Total

377847

21

,1 D F d e n o t e s t h e degrees o f f r e e d o m . ,2 F ' i s t h e r a t i o o f t h e m e a n square b e t w e e n l a b o r a t o r i e s t o t h e m e a n s q u a r e w i t h i n laboratories.

variations of analytical results within + 25% of the mean for total PCB c o n t e n t is not an unreasonable range for marine samples. The results of a oneway analysis Of variance (BMD07V: Dixon, 1971), are shown in Table IV. The value of F indicates how the means are related. For the 95% confidence level, F = 3.02 (Crow et al., 1960). For this work F = 28.8, indicating at least one of the means is significantly different. Several factors were the main contributors to the variability.

Methodology Although it appears that the techniques adopted by each laboratory are similar(Table V), identification of specific errors is difficult since it is virtually impossible to monitor processing of samples. However, u p o n closer inspection and analysis of the data, some general trends are apparent. An increase of Z PCB w i t h increasing dry weight of sediment is shown by a least~square treatment of the data (Fig.l). The curve has a slope of 1.35 and a correlation coefficient of 0.21. A plot of Z PCB vs. wet weight of sediment

159

(Fig.2) shows a similar behavior with a slope of 0.43 and a correlation coefficient of 0.11. The large scatter of the data and the absence of obvious trends suggest that the efficiency of recovery is not a function of sample size. I000

J 800

[]

600 J

81

4OO

200

2'o

~o

~o

~o

i~o

i~o

Fig.1. Plots of total chlorohiphenyl concentrations (ppb dry weight) vs dry weight of sediment(g). A A, O, B; • , C; ~ , D; A, E; O, F; ~ , G, ~ , H; 0, I ; a n d O , J. T h e i n n e r line is the least squares plot and the outer lines represent the 95% confidence limits. The encircled points are from extraction of dry sediments.

I000

800

600 @ a.

O

40O

200

20

40 WET

60 80 WEIGHT (g)

100

1:;'0

Fig.2. Plots of total chlorobiphenyl concentrations (ppb dry weight) vs wet weight of sediment (g). A A; ~ , B; • , C; @, D; A, E; O, F; ~ , G; ~, H; and 0, I. The inner line is the least squares plot and the outer lines represent the 95% confidence limits, The encircled points are from extraction of dry sediments.

160

The data was reduced ~further and separated into two groups; the ones in which dried or partially dried sediment was extracted (Laboratories A, C, F and J, encircled in Fig.l) and those in which the sediment was extracted wet. From a statistical t-test, significant differences between the t w o groups were apparent at the 95% confidence level ( t x p t = 3.75 vs. t0.0~s,2o = 2.81), with laboratories A, C, F and J reporting higher recoveries. It .should be noted here, that we have confirmed this b y performing an additional analysis of 1 g of sediment which yielded quantities within 10% o f the values determined b y A, C, F and J. A t-test performed on the data has shown no differences at the 95% confidence level ( t e x p = 0 . 8 8 ) , between laboratories that used acid cleanup and those that used column chromatography. The Soxhlet method of extraction, in which a variety of solvents can be used, was not evaluated in this study.

Sample inhomogeneity The existence of gradients among aliquots that might have been induced b y incomplete mixing of the initial sample was tested b y plotting Z PCB vs. % solids (Fig.3). No systematic trends were apparent. tO00

I

I

I

I

800

600

~•

B r'l

0

@

o_ 400

O

200

1

I

40 %

_

I

60

I

80

I00

SOLIDS

Fig.3. Plots of total chlorobiphenyl concentrations versus % solids. 4, A; o, B; e, C; @, D; a, E; o , F; ~, G; ~1, H; <), I; and O, J .

Spectral analysis The total chlorobiphenyl concentrations were reported in three forms: (1) w i t h o u t c o m p o n e n t identification (I), (2) with components identified as members of the Aroclor series (II), and (3) with components identified as homologs (III). The corresponding designations for each laboratory are shown in Table V.

freeze dry

thaw

thaw, suction filtration thaw

NR freeze dry

thaw

thaw

thaw steam dried

A

B

C

D

E F

G

H

I J

*Reported as Aroclor 1254 only. NR, None reported.

Pre-extraction

Laboratory

Soxhlet Soxhlet (2 : 1 acetone/hexane) homogenizer, boil (w/1:1 isopropanol/benzene) ultrasonic homogenizer (acetone) Soxhlet (acetone) Soxhlet (hexane)

Florisil,alumina, silicicacid Florisil,Hg Florisil,Hg

Hg, alumina, silicicacid Cu, alumina, silicagel FIorisil,Hg Cu, Florisil Florisil

I-Ig,H2SO 4

H~S04

Soxhlet (100% hexane) shake extraction ( 1:1 aeetone/hexane) Soxhlet ( 2 : 1 acetone/hexane) Soxhlet

Clean-up

Extraction

Summary of the methodology by each laboratory

TABLE V

6.4 5.49

66.2

NR

11.4 60.0

9.8

42.0

32.2

19.4

10.9 NR

112.3

10.4

20.0 103.8

27.4

57.9

45.7

33.8

Average weight of sediment analyzed (g) dry wet

II II

II

II*

II, III

I

II*

II

III

II

Spectral analysis form

O~

162

The first two forms include integrated spectral comparisons with Aroclor standards. The choice of the standard is usually arbitrary; some laboratories use a commercially available mixture, e.g., Aroclor 1254, with the sample content designated as such, while others utilize an o p t i m u m standard according to how well it matches the sample spectrum. In both cases, failure to account for internal c o m p o n e n t changes of the sample spectrum results in either an overestimate or an underestimate of the measured quantity depending on the spectral regime selected as the basis for comparisons. However, quantitation by homolog analysis (Willis a~nd Addison, 1972; Webb and McCall, 1972; Ugawa et al, 1973; Webb and McCall, 1973; Hirwe et al, 1974; Huntzinger et al, 1974) alleviates this effect since it is based on the actual chlorobiphenyl distribution occurring in a given sample, irrespective of the mixture's origin. SUMMARY

Assessment of CH concentrations in marine sediments depends upon methodology and spectral analysis techniques. Preextraction handling of the sample significantly affects the results, showing that extraction of dry sediments yields the highest recovery while differences in cleanup procedures did not influence the data. Although PCB's were measured by three different methods, the homolog approach can account for the changes of individual components in the sediment. To obtain the best estimate, it is recommended that (1) samples should be dried prior to solvent extraction and (2) results should be reported as homolog components. ACKNOWLEDGEMENTS

The authors acknowledge the support of the U.S. Environmental Protection Agency under Grant No. R-800362 for this work. We are also indebted to all participants for making this study possible. Acknowledgment for financial support is given also to NSF-IDOE by Professor Giam, Dr. Risebrough and Dr. Harvey. REFERENCES

Crow, E. L., Davis, F. A. and Maxfieid, M. W., 1960. Statistics Manual. Dover Publications, New York, N.Y., table 4. Dixon, W. J., 1971. BMD: Biomedical Computer Programs. University of California Press, Berkeley, Calif. Harvey, G. R., Miklas, H. P., Bowen, V. T. and Steinhauer, W. G., 1974. Observations on the distribution of chlorinated hydrocarbons in Atlantic Ocean organisms. J. Mar. Res., 32(2): 103--118. Hirwe, S. N., Borchard, R. E., Hansen, L. G. and Metcalf, R. L., 1974. Gas liquid chromotography - - mass spectrometric characterization of Aroclor 1242 components. Bull. Environ. Covitam. Toxic., 12(2): 138--143.

163 Holden, A. V., 1973. International cooperative study of organochlorine and mercury residues in wildlife, 1969--1971. Pestic. Monit. J., 7(1): 37--52. Horn, W. and Pavlou, S. P., 1975. Interlaboratory calibration study for assessing data reliability in chlorinated hydrocarbon analysis. Dept. of Oceanography, Univ. of Washington, Seattle, Wash., Spec. Rep. No. 59, Ref. No. M7537. Hutzinger, O., Safe, S. and Zitko, V., 1974. The chemistry of PCB's. CRC Press, Cleveland, Ohio, 44128; Ch. 2, 3, 13. Rote, J. W. and Murphy, P. G., 1971. A method for quantification of polychlorinated biphenyls (PCB) isomers. Bull. Environ. Contain. Toxic. 6(4): 377--384. Ugawa, M., Nakamura, A0 and Kashimoto, T., 1973. Calculation method for polychlorinated biphenyl (PCB) isomers in gas chromotographic determinations. Shokuhin Eiseigaku Zasshi, 14(5): 415--425. Webb, R. G. and McCall, A. C., 1972. Identities of polychlorinated biphenyl isomers in Aroclors. J. Assoc. Off. Anal. Chem., 55(4): 746--752. Webb, R. G. and McCall, A. C., 1973. Quantitative standards for electron capture gas chromotography. J. Chrom. Sci., 11: 366--372. Willis, D. E. and Addison, R. F., 1972. Identification and estimation of the major components of a commercial polychlorinated biphenyl mixture, Aroclor 1221. J. Fish. Res. Board Can., 29(5): 592--595.