THE DETERMINATION OF TRACES OF DIELDRIN IN BLOOD” J. G. REYNOLDS
The Shell Petroleum Company, Limited, Research Laboratories, Sittingbourne, Kent, England INTRODUCTION
Stringent conditions with regard to clean- liness are laid down for the attention of public authorities concerned with the spray- ing of dieldrin formulations. Protective clothing must be worn and this must be changed frequently. Personal cleanliness is imperative. Since sprayers are often operat- ing in rugged country for days at a time it is not easy for these conditions to be fol- lowed and it has been suggested that analy- sis of the blood of operators might revea1 traces of dieldrin which would enable authorities to take precautionary measures. Little detail is available in the literature concerning the analysis of blood for dieldrin. In fact, the only evidente we have is from a private communication and concerns the determination of aldrin and dieldrin residues in the blood of cattle, following subcutaneous injection of these insecticides. In these determinations blood samples (400 ml. each) were analyzed, at intervals after injection, by bioassay and colorimetric procedures. The sensitivity of these methods is quoted as being 0.02 ppm to 0.03 ppm for the bioassay (i.e. on 400 ml. 8 to 12 ,ug of toxi- cant) and 0.02 to 0.05 ppm for the colori- metric procedure (i.e. on 400 ml. 8 to 20 pg of toxicant). Recoveries of dieldrin added to uncontaminated blood in order to produce a concentration of 0.1 ppm (i.e. on 400 ml. 40 pg of toxicant) are reported in this private communication as being about 50% by both methods.
THE ANALYTICAL METHODS
As a basis for our investigations we had available supplies of human blood from the * Paper presented at the Symposium on Toxi- cological Investigations of Dieldrin in Venezuela, sponsored by the Division of Malariology of the Ministry of Public Health and Social Welfare, and held in Maracay, Aragua, Venezuela, May 15-16, 1957.
blood bank of the British Transfusion Service, a method for the chemical determi- nation of dieldrin in crep materials (1) and a method for the determination of aldrin (2). Briefly, the procedure consisted of sapon- ifying aliquots of blood with alcoholic potash, extracting the saponified solution of blood with hexane, chromatographing the solution through magnesia/Celite and ana- lyzing the main bulk of the “clean up” fractions by the colorimetric reduction phenyl azide method, a smaller portion being reserved for bioassay, if necessary, using Drosophila melanogaster. It is necessary to calibrate the magnesia/Celite absorbent using a saponified blood solution to which a known concentration of dieldrin has been added. It is also necessary to add dieldrin to blood aliquots, prior to saponification, at different concentration levels, calculated to cover the range in which the dieldrin con- centration in the spray operators’ blood is expected to fall, and to replicate each con- centration to ascertain the precision of recovery. The assumption must be made that the extraction of any dieldrin from the blood of spray operators would parallel that of dieldrin added to the blood of uncontami- nated controls.
We do not propose to cover precise de- tails of the whole “clean up” procedure and colorimetric finish since these are dealt with very adequately in the publication by our colleagues, O’Donnell, Johnson and Weiss (1). We would, however, Iike to emphasize some points in the published method which would make it more easily applicable to blood (as distinct from crep) analysis.
528 BOLETIN DE LA OFICINA SANITARIA PANAMERICANA
loss. However, if the concentration of dieldrin in the sample blood is expected to be low and if the patient from whom the blood is taken is available as a reserve, we would advise analysis on the whole sample divided into two replicates thus ensuring, in the colorimetric finish, that the excess of color due to the sample blood relative to that due to the control blood is at a maxi- mum.
2. The instructions given in the genera1 method (1) for saponification of samples contaimng glycerides have been amplified for blood analysis in Appendix 1.
3. After saponification the solutions are extracted with purified extraction solvent and give yellow to brown colored extracts. The importance of the solvent purification cannot be over emphasized, the real criterion being, not that it should have a certain boiling point or residue on evaporation, but that 500 ml. of it should give color in the colorimetric finish of the method equivalent to a net optical density (1 cm. cell) of not more than 0.05, representing about 4 pg of apparent dieldrin. If this figure is exceeded, further purification should be considered.
4. The yellow colored extracts are now chromatographed through columns of mag- nesia/Celite mixture. The general behavior of a solvent extract of saponified blood to which a known concentration of dieldrin has previously been added must first have been studied before the “clean up” of sample blood extracts is attempted. It is essentía that the procedure for preparing the absor- bent, filling the columns and chromatogra- nhing the sample extracts should be identical in every detail to that found to be success- ful during this calibration of the absorbent mixture. Since the pigment to dieldrin ratio in any blood sample is bound to be high, the effect of the non-removal of even 1% of the interfering compounds, on the colorimetric finish, can well be imagined even though, weight for weight, the dieldrin has much the greater color-forming potential. The “cleaned up” extracts must be colorless.
At this stage it might be considered
desirable to carry out a bioassay. Details of a method in common use in our laboratories are given in Appendix II.
5. After chromatography, these extracts containing al1 the dieldrin present in the original blood aliquot are finally evaporated slowly to dryness in a gentle stream of dry air. When a large number of extracts are being evaporated the volume of each is not, in our experience, reduced at the same rate and constant vigilance is needed to ensure that each solution is taken just to dryness. While we do not think that any dieldrin is lost during the evaporation procedure, it will certainly be lost if air blowing is con- tinued after the removal of the last traces of solvent.
6. The “cleaned up” dieldrin residues are now refluxed with a mixture of hydrobromic acid and acetic anhydride and then redueed with zinc dust to produce a compound very similar in chemical structure to aldrin. The acid mixture after refluxing, but prior to the addition of zinc must be no more than faintly brown. If darker colors are obtained at this stage, these particular analyses must be abandoned since high blanks and “off colors” in the colorimetric finish are almost inevitable.
Lkember 19571 TOXICOLOGICAL INVESTIGATIONS OF DIELDRIN 529
filtering the f?nal colored solutions. Failure
to remove the waxy suspension prior to the of ,blood Voluta
addition of the diazotised dinitro-aniline has a;;q;ts been shown to interfere with the color -
formation. 20
EXPERIENCE WITH THE ANALYTICAL METHOD 20
The recovery of dieldrin from blood samples to which dieldrin has been added is shown in the table on this page.
The results are net, after deduction of
the blank obtained from 20 ml. of blood 2o to which dieldrin has not been added.
In conclusion we hope we have not painted too gloomy a picture of the difhculties entailed in this analysis. Using the published methods and taking into account the special sources of error described in this paper, we 20 feel that any reasonably equipped laboratory
could quite quickly produce reliable results. -
REFERENCES
- 1 -
-
Xeldrin added
i -
l
-
0 PPm 0.25 05 1.0 20 -I i .-
-
Dieldrin recovered :n Pg A
5 5 7 13 12 ll II 1 28 15 ll 22' 12 14 8, 16 391 50/
-
I
44--
Dieldrin recovered
- in ppm 0 25
1 0.25 0 35 0 65 0 60 i 0 55 0 55 i 14 0 75 0 55 1 11 t 06 07 04 I
1 95 2.5
--_ .verage - .- 0 25
0 55
0 80
(1) O’Donnell, A. E.; Johnson, Jr., H. W., and (2) O’Donnell, A. E.; Neal, M. M.; Weiss, F. T.; Weiss, F. T.: Jour. Agr. Food Chem. 3, 757, Bann, J. M.; De-Cino, T. J., and Lau, S. C.:
1955. Ibid 2, 573, 1954.
Appendix 1
PREPARATION OF BLOOD SAMPLES FOR THE COLORIMETRIC DETERMINATION OF INSECTICIDE RESIDUES
The blood sample is weighed into a suitable conical flask. Although large samples are ad- visable, particularly when it is suspected that the dieldrin concentration will be low, samples larger than 100 g. are inconvenient to handle. To the flask is added 0.5 ml. of 50% aqueous potassium hydroxide and 3 ml. of 95% ethanol for each g. of blood, using a minimum of 3 ml.
of potassium hydroxide and 18 ml. of ethanol. The mixture is refluxed gently on a steam bath for two hours. On removal from the steam bath, a volume of distilled water equal to one half the volume of alcohol used is added through the top of the reflux condenser. The mixture is cooled and transferred to a l-2 litre separating funnel using 10 to 15 ml. portions of ethanol to effect the transfer. A volume of purified extraction solvent (hexane) equal to that of the saponifica- tion mixture is added. The mixture is shaken vigorously, the layers allowed to separate and
530 BOLETIN DE LA OFICINA SANITARIA PANAMERICANA
Appendix II
BlOASSAY OF BLOOD EXTRACTS AFTER SAPONIFICATION AND CHROMATOGRAPHY
One ml. of the “cleaned up” extract (equiva- lent to a known volume of blood) is pipetted evenly onto the surface of a filter paper (15 x 6.5 cms.). After allowing the solvent to evapo- rate, the paper is enclosed in an open ended glass tube (8” x 1”).
A standard food medium, as used for the cul- ture of the test insect, is placed in one end of the tube. Approximately 40 Drosophila melano- gaster of mixed sexes up to 24 hours old are then introduced into the tube and the ends covered
with muslin. A standard series using solutions of toxicant in the extract from uncontaminated blood is made up for comparison.
