Navy Chemicals in Potable Water 2

8/9/2019 Navy Chemicals in Potable Water 2

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DIOXIN

What the U.S. Navy knew and didnt or wouldnt tell us.

An OP-ED Paper by Chuck Graham

Im a U.S. Navy Vietnam Veteran and I have had a claim in place with the Dept of Veterans

Affairs since 2003. Like so many of you Ive been on the hamster wheel and suffered throughthe Haas appeal all to no avail. Over the years Ive researched any available material that

might help prove that the U.S. Navy had knowledge to support the findings of the Australian

study, {ENTOX, also called NRCET from 2002}. This study involved the co- distillation of

Dioxin through the fresh water evaporator systems commonly used aboard RAN, Royal

Australian Naval Ships, that were present in Vietnam. The same evaporator systems were

commonly used by U.S. Navy Ships, that were present in Vietnam, as the majority of the

Australian Naval Ships were built at U.S. Naval Shipyards. It is my hope that the following

information will shed some knowledge of what the U.S. Navy knew and had in their

possession and if they knew then the DOD and more than likely the DVA also had to know.{ My co-investigator on this paper is Ms Susie Belanger, known to many veterans across the

country and without her help this would not have been possible.}

As far back as 1946 the U.S. Navy had knowledge of the dangers of distilling water for

shipboard uses while in littoral waters or certain other locations. This was evidenced by the

fact that while conducting atomic radiation testing at Bikini Atoll, they were warned not to

utilize any seawater aboard ships in the area, for fear of contamination by the radiation which

had contaminated the coastal waters. This was Operation Crossroads and 79 ships that were

present during these tests, were salvaged and sent to Hunters Point Naval Shipyard in San

Francisco for decontamination. An acid wash had to be used to decontaminate the evaporatorsand water purification systems.

In the U.S. Army Technical Manual TM 5-813-8 from September 1986 on water

Desalination chapter 5-1 paragraph C where it states that dissolved organic materials will

carry across a distillation / condensation process with the water. Pesticides and industrial

organic chemicals may be difficult to remove by distillation/condensation.

Ok folks, lets look at and re-read that statement !! Someone in the Army had to have done

some tests to make that statement. How else would they have known, without testing the

condensate, that this was so. That proves that the Military knew that dioxin/

herbicides/pesticides would remain in distilled water.

The manual of Naval Preventive Medicine {NAVMED P -5010-6 rev 1990} chapter 6 Water

Supply Afloat sec 6-3 states That water in harbors or off shore from habitations and when

operating in fleet strength must be considered polluted and unfit for uses other than in

fire and flushing systems and must not be used for other purposes.

In the U.S. Navys Risk Analysis of Shipboard Drinking Water Chemical Contaminants,

August 18, 2000 by Lieutenant Michael D. Cassady Medical Service Corps U.S. Navy


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states An important aspect of the drinking water produced onboard ships and submarines is,

its source. Ships and submarines routinely do not produce water unless they are at least 12

miles from the shoreline. However, the operational environment for ships and submarines is

changing and more missions are requiring operations in littoral waters for extended lengths of

time. Littoral waters are more likely to be at risk for primary and secondary contaminates.

Now while on the gun-line conducting NGS firing missions off the coast of Vietnam, we didnot have time to pull off and run out 12 miles and make fresh water. We made water where we

were at, 24/7

Now to get to the heart of the matter and the reason for this paper. We have discovered

several Naval Documents that we feel should shed some light on the knowledge that the U.S.

Navy had over the years starting in 1963 with BUMED INSTRUCTION 6240.3B from

Chief, Bureau of Medicine and Surgery to ALL Ships and Stations. Subject : Standards for

Potable Water. Purpose , to establish standards for water for drinking and culinary purposes

throughout the Naval Establishment.

Pay special attention to page 3 where it lists Chemical Characteristics Limits Nowhere doyou see Herbicides mentioned.

[Editor's Note: OK... here's a smoking gun. In 1963, water treatment specifications did not

mention insecticides/herbicides in the water. In 1972, these same water treatment specifications

mentioned insecticides/herbicides needing to be addressed. If they didn't know about the presence

of herbicides in the water, why would that be specifically called out? Someone wearing brass

and a lot of gold stripes lied to the American public. I wonder who that was?]


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Then in 1972 we see BUMED INSTRUCTION 6240.3C From: Chief, Bureau of Medicine and

Surgery To : ALL Ships and Stations Subj: Standards for Potable Water.

Pay special attention to page 6 on Chemical Concentrations where it now includes Pesticides, Herbicides,

Fungicides and see footnote {2} This is just a short period of 9 years ,1963 through 1972 that something

brought to their attention that it would be desirable to remove Pesticides and Herbicides from our drinking

water. In my humble opinion scientific tests of some sort had to be conducted to verify this concern over

Herbicides.


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DEPARTMENT OF THE NAVY BUJ~EDiNS-i- 624~.X/G-l-1.Bureau of Medicine and Surgery 722.PAT: cb -Washington, D.C. 20390 13 December 1972

.

BUMED INSTRUCTJON 6240.XCHANGE TRANSMITTAL 1Chief, Bureau of Medicine and Surgeryrom:To: All Ships and Stations

Subj: Standards for potable wafer

1. Purpose. To promulgate change 1 io the basicinstruction io eliminate possible confusion concern-inghow titrate and titri~e levels are to be determined.

These levels arc IO be expressed as nitrate nitrogenor nittite nitrogen which is in consonance with ;CUirent testing procedures. I1.II2. Action. On pagq 4, table, line 12 , opposite entr)r !for Nitrate and Nitrjte, in the Concentration column,;to present 10. add (as N) so rhar it will read:

10. (as N)

G. M. DAVISDistribution:SfGt--PafiGa~dZMARCORPS Code CC (less MarBks)Stocked:COMNAVDIST WASH DC(Supply & fiscal Dept.-Code 514.3)Wash. Navy Ya:dWash., D.C. 20390.-

-I . .;.CLW .

0000000144c, ( ), . .


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,. .

x DEPARTMENT OF THE NAVY BUMEDINST 624CL3Ci Bureau of Medicine and Surgery 722-PAT: cbWashington, D.C. 20390 25 August 7 972

BUMED INSTRUCTION 6240.3C. From: Chief, Bureau of Medicine and SurgeryTo: AiI Ships and Stations

Subj: Standards for potable water* Ref: (a) NAVMATINST 5711.9A of 17 June* 1965 (NOTAL): . * lb) EUMEDINST 5711.2A of 3 December+ 1965

. 1- Purpose. To kstablish standards-for waler for.* .row a.nd culinarymoses throughout the Naval+ Establishment and prescrjbe the use of the DD Form* 686, Bacteriological Examinarion of Water, and DD* Form 710, Physical and Chemical Analysis oi Water.* 2. Cancellation. BUh4ED Instructions 6240.313 and* 6240.5 are canceled.

3, Backgrounda. Policy. The Departmenr of, Defense has

establiskd the policy of compliance by tie MilitaryDepartments with United States public Health ServiceDrinking Wqter Standards, as may be modified by theMedical Se&ices of the Departments, or as may bemodified by competent authority for purposes ofinternational agreement.

b. Internatio~ai Agreement. Naval TripartiteStandardization Agreement ABC-NAVY-STD-23Awas promulgated by references (a) and (b). The objectof the agreement is to provide the United States Navy,the Royal NG&$y*&d the Royal CanadGn Navy assur- : .ante that drinking and culinary water delivered to&%I others ships from installations under their cog-nizance meet? certain ,minimum standards of quality.4. Quality Standards. The standards for bacteriologi-cal quality, physical and chemicJ charac teristics, andradioactivity shall be those in Public Health ServiceDr5&ing Water Standards, 1962 Department ofHealth, Education, and Welfare. The Standards, asmodified, may be found in NAVMED P-5010-5, WaterSupply Ashore, available through the Navy SupplySystem.,,w..: ,I

5. Definition of Term:. The following terms a:rc he-fined for clarification in interpretation of standards:

a. Adequate protection by natural means invblvesone or more 0E the following processes o f nature.lhatproduce water consjstcntly meeting the requirementsof these standards: dilution, storage, sedimenl.atjon,sunlight, aeration, and the associated physjcal andbiolo&cal p rocesses which tend to accompljsh naturalpurification in surface waters and, in the cast ofground waters, the natural purilicarion of water byinfiltralion through soil and percolation tkTOU$underlying material and storage below the groundwater t2bk.

b. Adcquatc protection by treatment rncans anyone or any combination bf the c.onrrolled processesof coagulation, sedimentation, absorption, fjltratjon,disinfection, or other proccsscs which product: awater consistently meeting the rcquiremcn tS of thesestandards. This protection also includes processesWlljcil arc appropriate lo the source of supply; workswhich arc of adequate capacily to meet maximumdemands without creating hcaith hazards, and whichare located, designed, and constructed to clirmnate orprevent pollution; and conscicnLious operation bywell trained and competent personnel whose qualifi-cations are commensurate with the responsibilities oithe position.

c The coliform group includes all organisms con-sidered in the coliforrn group as set forth in Standard-Methods for the Examination of Water and Wastewa ter,current edition, prepared and pub&shed j oti Lly by the . ..yT.2 >::I ._, ..Amerjcan Public fiealth Association, American Wattr ?:.i% . Works Association, and Water Pollution Contiol . .Federation.

d. Health hazards mean any conditions, devices, ,orpractices in the water supply system and its operationwhich create, or may create, a danger to the healthand well-being of the water consumer. An example ofa health hazard is a structural defec t in the watersupply system, whether of location, design, or con-struction, which may IegulaIly or occasionally prevent

0000000145 i -)i _.--- -- - .--- - .~--- ._--_ ---_ -- -.. - - -- - - - -- - -------_-~____- ---.... - - -~- _____ _. _~ ___ - _- - - .-. . _ - - . - .. .- - - .. 2. .- -__ .=-- VI ?.


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CUMEDINST 6240.3C25 Augur 1972 q26: Pollution, 2s used in these standards, me,ans

the presence of any foreign substance (organic, in-organic, radiolo@& or biological) in water whichtends to degrade its quality so as to constitute ahazard or impair the usefulness of the water.

f. The standard sample for the bacteriologicaltest shall cons:s~ oi:

(11 For the bacteriological fermentatjon tubetest, live standard portions of either:

(al 10 milliliiers(bl 100 milliliters ~.--~__~

(21 Fcjr the membrane filter technique, notless than SO milliiiters.g- Uater supply system includes the works and a. Bacteriological Quality (Limits). The presenceauxiliaries for collection, treatment, storage, and dis of organisms of ihe coliform group as indicated bytribution of the water from the sources of supply to samples examined shall not exceed the following-the freeflowing outlet of the ultimate consumer. limits:

6. Source and Proyectiona. The water supply should be obtained from the

most desirable source which is feasible, 2nd effortshould be made to$vent or control pollution ofthe source. If the source is not adequately protectedby titural means, the suppiy shall be adequatelyprotected by treatment.

b. Frequent saniary sweys shall be made of thewater supply system to locate and identify healthhazards which might exist in the system.

qualifications are acceptable to the Navy Facilities * :En:@neering Command or Navy Ship Systems Corn- * :mand. c I

(41 Adcquatc capacity to meet peal: demands iwithout development of Jaw presstires or other health ihazards; and /!

(5) Record of laboratory examinations show-ing consistem compliance with ihe water quality re.,quiremenfs oi these standards. I

7. Srandards. The limits ljsted below arc generallythose contained in Public Health Service Drinking

-TEfEFSt~fii~~ds, ~9~~~~r-s~m~~~~~ced~s~nl~-- -----~ _~~tcchniqucs, refer to NAVMED I-5010-S.

\(I 1 When IO ml. slandarcl portions are cx-amined, not more than i0 pcrccnt in any month shailshow the presence of the coliforn~group. The presenceof the coliform group in three or more 10 ml. portionsof a standard sample shall not be allowable if thisoccu.rs: I

(a) In two consecutive samples;(b) In more than one sample per month

when less than 20 are examined per month; or(cl In mom than fin percent of the samples

c .4ppronl of water supplies shall be dependentin part upon:

(11 Enforcement of rules and regulations toprevent development of health hazards;(21 Adequate protection of the water qualitythroughout all parts of the system, as demonstratedby frequent surveys;I31 Proper operation of the water supply systemunder the responsible charge of personnel whose

When organisms of the coliform group OCCUTn threeor more of the IO ml. portions of a single standardsample, daily sampies from the same sampling pointShari be coLlected promptly and kxamined until theresults obtained from at least two consecutive samplesshow the water to be of satisfactory quality.

(21 When 100 mkstandard porTions are ex-amined, not more than 60 percent in any month shallshow tie presence of the coliww. The presence

0000000146


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33UMEDINST 6240.3C25 Augus 197.2

.. .of the coliform group in all five of the 100 ml. por-

tions of a standard sample shall not be allowable ifthis occurs:, (a) In two consecutive samples;

(b) In more than one sample per monthwhen less than five are examined per month; or

(4 In more than 20 percent of thesamples when five or more are examined per month.?4hen organisms of the coliform group occur in allfive of the 100 ml. portions of a singglc standardsample, daily samples from the same sampling pointshall be collected promptly and examined until the---..--------- ---se~~t5~b?-a~e~~~~st~ _conse~~t&SaXgle~show the water to be of satisfactory quality.

(3) When the membrane filter technique isused, the arithmetic mean coliiorm density of allstandard samples examined per month shall notexceed one per 100 ml. Coliform colonies perc. standard sample shall not exceed 3/50 ml., e/100 ml.,

/ 7/200 ml., or 13/500 ml. in:(a) Two consec::tive samples;: .(b) More than one standard sample when

less than 20 are examined per month; or(d More than five percent of the standardsamples when 20 or more are examined per month.

When coliform colonies in asin$le standard sampleexceed the above values, daily samples from rhc samesampltirg point shall be collected promptly andexamined until the results obtained from at least twoconsecutive samples show the water to be of satisfac-tory quality. .

b. Bacteriological Examination of Wate r. Bacte- +riological Examination of Wate r, DD Form 686, shal)be used by all naval facilities, both ashore and afloat,to conduct bacteriological exzunination of water.

c. Physical Characteristics (Limits). Drinkingwater should contain no impurity which would causeoffense to the sense of sight, taste,orsnle~~~J~iaer--.----------~-----general use, the following limits should not be ex-ceeded:

Turbidity. . . . _ _ . . . _ . . . _ . . 5 unitsCDlOr. . . _ . . . _ . . . . . . . . . _ _ . 15 unitsThreshold Odor Number. . . _ _ 3

d. Chemical Characteristics (Limits). Drinkingwater shall not contain impurities in concentrarionswhichmay be hazardous to the health o f the con-sumers. It should not be excessively corrosive to thewater supply system. Substances used in its treatmenr

f---*, .t CLW .\.. %: Q~.uuml I._--- -------.---.~--.-------------.---;.. .--.-. -...-..--..--.-..^ ..-, _ -.


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BUMEDlNST 6240.31:25 August .7972shall not remain in the water in eancentrations (1) The following chemical substances shouldgreater than required by good practice. Substances not be present in a water supply ill excess of the listedwhich may have deleterious physiological effect, or concentrations ~vhcre, n the judgcment of the Navyfor which physiological effects are not known, shall Facilities Engineering Command and the Bureau ofnot be introduced into the system in a manner which Medicine and Surgery, other more suitable supplieswould permit them to reach the consumer. are or can be made available.

SubstanceConcentration .~J-J dl (pv)

Antimony (Sb) (See footnote 1.). ................................................ 0.01Arsenic(As). .................................................................. 0.01Ch.loride(CI). ............................................................... 250.~~r~~nChi-rn~~~~~~~~ ;-:-Y-.;- --:--: Tx- -----c;T-.--z .-~__--_.-_. .. .-. ._-. ____ --.._ I _.s._._l_.L!.JJ. ___ ._ -., .. ._.____....... __. _Copper(Cu)..........................-......-...........-.......~......~~- .. 1.Cyanidc(CN)..........................-.........-......................- .... 0.01Fluoride(F)............................-........--.......~........: ......... ee 7d(3)Iron ................................................................... 257ManganeseO\4n)...................................-....-- .................... 0.05Mercury(Hg)(Seefoornote2.). ................................................. 0.005Methylene Blue-Active Substance (Including A%) .c................................... 0.5Nitrate (NOa), Nitrite (NO?) (See footnote 3.) c....................................... 10.p~(Range)...................-.........-...-....-............-..-......- .... 6.0 - 9.0 + --.\;Phenols .................................................................... 0.001Sulfate(S04)..:.. ............................................................. 250.Total Dissolved Solids ......................................................... 500.ZINC(Zn).* ...... It11

................. l.... .................................... 5.

Footnotes:1. Not contained in Dxinlcing Water Standards but this limit set by PHS and BUMED.

I2. Not contained in Drinking Water Standards but this limit set by BUMED upon recommendation.of EPA. I3. In areas in which the nitrate or nitrite content of water is known to bem excess of the listed concentration, *

,, the public should be warned of the potential dangers of using the water for infant.feeding. I1

I


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..- -.__. L

/ - >I BUMEDINST 6240.3C-__ 25 August 1972.-

(2) The presence of the fol3owing substances m excess of the concentrations listed shall corqtjiutegrounds for rejection of the supply:

Substance Concentration~JIw/l bpm>Antimony (Sb) (See footnote 1.) ................................................Arsenic (As). ................................................................Barium(Ba). ................................................................Cadmium (Cd) ................................. .. .............................Chromium (Hexavalent) (Cr6). ........ : ........................................Cyanide(CN). ..................................................... :....._ ...Fluoride(F)...:........................:....................~.-......~..~ ...Lead(Pb) ..................................................................

-Pesticides;%erbacides;Fungicities-(See footnote2.)Chlorinated hydrocarbons ....................................................Organo-phosphates ..........................................................Chlorophenoxy herbacides ...................................................Selenium (Se). ...............................................................S~ver!Ag).......................................................-- .........

0.050.05

..00.01o-o.50.2See 7d(3)0.05 s0.003 - 0.10.10.005 - I .oo0.010.05

Footnotes:7. Not contamed in Dtin!~kg Water Standards but this limit set by PHS and BUMED.:2. Concentrations represent range of levels for each group of chemicals. Individual pesticides have specific con-centrations ~Q~eries should be directed to BUMED (Code 72).

(3) huoride. When fluoride is naturally present values in the table shall constitute grounds for rejec-m drinking water, the concentration should not aver- tion of the supply. Men fluoridation (supplementa-age more than the appropriate upper limit in the tion o f fluoride in drinking wate$ is practiced, thefollowing table. Presence of fluoride in average con- average luoride concentration shaI.l be kept withincentrations greater than two times the optimum the upper and lower control limits in the table.Annual average of m2ximu.ilIdaily air temperatures, basedon data obtained for a minimumof 5 years I Lower Optimum I Upper

09 1.2 1.70.8 1.1 1.50.8 1.0 1.30.7 0.9 1.20.7 ..0.8 1.00.6 0.7 0.8,* e. Physical and .Chemical Analysis o f Water. Physicat be used by all naval,fa,cif-- + and Chemical hnalysis of Water, DD Form 7 IO, shaU to conduct phydcai and chemical analysis of water.

50.0 - 53.753.8 - 58.3.58.4 - 63.863.9 - 70.670.7 - 79.279.3 * 90.5

0000000 4 49 it

.( ,:.;r!


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BUMEDI NST 6240.3C25 August 1972

f. Radioactivity [Limits).

(7 1 The effects of human radiation exposureare viewed as harmful and. any unnecessary exposureto ioniztig radiation should be avoided. Approval oiwater supplies containing radioactive materials shallbe based upon the judgement that the radioactivityintake from such water supplies when added to thatfrom &I other sources is not likely to result in an j,n-take greater than th: radiation protection guidancerecommended by the Federal Radiation Council andapproved by the President. (The Federal RadiationCouncil; in its 13 September 1961, Memorandurn for.*:-trs$ient, recommended that Routine control ofuseful applications of radlatlon and atomic energyshould be such that expected average exposures ofsuitable samples of an exposed population group wiJlnot exceed the upper value of Range II{20 ppc/dayof Radium-226 and 200 WC/day of Strontium-90))Water supplies shzll be approved without further con-sideration of olher souices of raciioacrivity intake of

.-Radium-226 and Strontium-90 when the water con-tains these substances in amounts not exceeding 3and 10 WC/liter, respectively. When these concentra-tions art exceeded, a water supply shall be approvedby the certifying guthority if surveillance of total in- Itakes of radioactitir from all sources indicates thatsuch intakes are tii in

:- the limits recommended by ithe Federa l Radiation Council for control action. !.

(2) fn the known absence (taken here to mean La negligibly small fraction of the above 3pecific limits,where the limit for unidensed alpha emitters is

t&en as the listed Limit for Radium-226) ofStrontium-90 and alpha emitters , the water supply .isacceptable when the gross beta concentrations do notexceed 1,000 fi&liter. Gross beta concentraiions inexcess of 1,000 WC/liter shall be pounds for rejec-tion of supply except when more complete analysesindicate that concenrrations of nuclides are not like.lyto cause exposures greatej than the Radiation Protec-tion .Guidis as approved by the President on recom-mendation of the Federal Radiation Council. 28. Technical Assistance. Assistance with parable waterproblems may be requested from the followin.g:

in accordance with BI,TMED Instruction 6200.3Cseries, Subj: Environmental and Preventive IMedicineUnits.

b. Navy Facilities Engineering Commands FieldEngn~cenr~g OKices in :iccordsncc wiijl current ;


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Then in February 1987 we have the document from Naval Facilities Engineering Command

Guide Performance Work Statement {GPWS } For Water Plants and System Operation and

Maintenance. Prepared by Southern Division Naval Facilities Engineering Command in Charleston, South

Carolina.

It states The contractor shall produce and store treated water free of taste and or odor and that meets the

minimum water quality standards described below: see page 44 {PDF DOC } Where we see Herbicides are a

concern again.


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NAVAL FACILITIES ENGINEERING COMMAND

GUIDE PERFORMANCE WORK STATEMENT (GPWS)

FOR

WATER PLANTS AND SYSTEMS OPERATION AND MAINTENANCE

PREPARED BY

SOUTHERN DIVISION

NAVAL FACILITIES ENGINEERING COMMAND

2155 EAGLE DRIVE, P. O. BOX 190010

CHARLESTON, SOUTH CAROLINA 29419-9010

FEBRUARY l987


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shall produce sufficient potable water to meet demand up to a maximum of !INSERT NUMBER OF GALLONS! gallons per !INSERT TIMEFRAME!. The Contractor shallmaintain the system so that in the event of a draw down below the system'sminimum storage limit, storage volumes can be replaced at the rate of INSERTNUMBER OF GALLONS! gallons per !INSERT TIMEFRAME!. The Contractor shallproduce and store treated water free of taste and/or odor, and that meets theminimum water quality standards described below:

UNIT OF PERFORMANCEMEASURABLE OUTPUT MEASURE LEVEL (# OF UNITS)

INORGANIC CHEMICALS MAX. ALLOWABLEArsenic mg/l 0.05Barium mg/l 1.0Cadmium mg/l 0.010Chromium mg/l 0.05Lead mg/l 0.05Mercury mg/l 0.002Nitrate (as N) mg/l 10.0Selenium mg/l 0.01Silver mg/l 0.05

ORGANIC CHEMICALS MAX. ALLOWABLE

Endrin mg/l 0.0002Lindane mg/l 0.004Methoxychlor mg/l 0.10Toxaphene mg/l 0.0052, 4-D mg/l 0.102, 4, 5-TP Silvex mg/l 0.01TTHM mg/l 0.10

TURBIDITY NTU 1.0

MICROBIOLOGICAL NOTE (1) NOTE (1)(coliform)

(a) Membrane filter technique

or(b) Fermentation tubes with 10 ml.standard portions; 5 - tube MPN.

RADIOACTIVITYRadium 226 + Radium 228 p Ci/l 5.0Gross Alpha (NOTE 3) p Ci/l 15.0Beta particle/photon mrem/yr 4.0

CHLORINE RESIDUALSample point (a) mg/l 0.2(min. allowable)Sample point (b) mg/l 0.2Sample point (c) mg/l 0.2Sample point (d) mg/l 0.2Sample point (etc.) mg/l 0.2

FLUORIDE mg/l 0.7-1.2NOTE (2)

HARDNESS mg/l as CaCO3 180

NOTE (1): Obtain values from 40 CFR 141.14 and 141.21 for: (a) membranefilter technique and (b) fermentation tubes with 10 ml standardportions; 5-tube MPN.

C-10


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Finally, see the following study where Researchers in Vietnam in 1970 tested fish and crustaceans

For the presence of TCDD {Dioxin}. These are the same researchers that were mentioned in the

Australian ENTOX study and the fish tested were caught by local fishermen in Vietnam, both in

fresh water as well as saltwater. This shows that dioxins were present in local fish in 1970 and

If dioxin stopped at lands-end, as DVA would have us believe, how did it pollute saltwater fish

and crustaceans.


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An Analytical Method for DetectingTCDD ( D i o x i n ) : Levels o f TCDD i nSamples from Vietnam

b y R o b e r t B a u g h m a n * a n d M a t t h e w M e s e l s o n *2 , 3 , 7 , 8 - T e t r a c h l o r o d i b e n z o - p - d i o x i n ( T C -DD) i s a n e x t r a o r d i n a r i l y t o x i c s u b s t a n c et h a t i s p r o d u c e d a s a n u n w a n t e d s i d e p r o d u c ti n t h e i n d u s t r i a l s y n t h e s i s o f 2 , 4 , 5 - t r i -c h l o r o p h e n o l , a n i n t e r m e d i a t e i n t h e manu-

f a c t u r e o f t h e h e r b i c i d e 2 , 4 , 5 - t r i c h l o r o p h e n -o x y a c e t i c a c i d ( 2 , 4 , 5 - T ) ( 1 , 2 ) . B e c a u s e o f i t sc h e m i c a l s t a b i l i t y a n d i t s l i p o p h i l i c n a t u r e ,t h e p o s s i b i l i t y e x i s t s t h a t TCDD r e l e a s e di n t o t h e e n v i r o n m e n t c o u l d a c c u m u l a t e i nf o o d c h a i n s . A d i r e c t t e s t o f t h e p o s s i b i l i t yo f b i o l o g i c a l l y s i g n i f i c a n t a c c u m u l a t i o n i na n i m a l t i s s u e s r e q u i r e s a n a n a l y t i c a l m e t h o da b l e t o d e t e c t TCDD a t l e v e l s w e l l b e l o wt h o s e known t o b e t o x i c . T h e l ow e s t v a l u eknown f o r t h e l e t h a l d o s e o f TCDD i s t h a to b s e r v e d i n t h e g u i n e a p i g , f o r w h i c h t h es i n g l e o r a l d o s e L D 5 0 i s 6 0 0 p a r t s p e r t r i l l i o n( p p t ) b o d y w e i g h t ( 3 ) . A l l o w i n g f o r s u b -l e t h a l t o x i c e f f e c t s a n d p r o v i d i n g f o r a c o n -s e r v a t i v e m a r g i n o f s a f e t y , i t s e e m s d e s i r -a b l e t o h a v e a n a n a l y t i c a l s e n s i t i v i t y o f a tl e a s t 1 p p t . F o r a 1 - g s a m p l e t h i s m e a n s t h em e t h o d m u s t h a v e a s e n s i t i v i t y o f . a b o u t1 0 - 1 2 g o r 1 p i c o g r a m ( p g ) .T h e m o s t common m e t h o d f o r a n a l y z i n gc h l o r i n a t e d o r g a n i c c o m p o u n d s i n t i s s u es a m p l e s i s g a s - l i q u i d c h r o m a t o g r a p h y ( G L C )w i t h a n e l e c t r o n c a p t u r e d e t e c t o r . I t s l i m i t

* H a r v a r d U n i v e r s i t y , C a m b r i d g e , M a s s a c h u s e t t s0 2 1 3 8 .

o f d e t e c t i o n f o r TCDD, a b o u t 1 0 - l 0 g , i s i n -a d e q u a t e . T h i s m e t h o d i s a l s o s u s c e p t i b l e t oi n t e r f e r e n c e f r o m o t h e r c o m p o u n d s a n d s oi s no t v e r y s p e c i f i c .M a s s s p e c t r o m e t r y o f f e r s b e t t e r p o s s i b i l i -t i e s . I t i s h i g h s e n s i t i v e a n d i n t h e h i g hr e s o l u t i o n m o d e o f o p e r a t i o n i t i s h i g h l ys p e c i f i c . We h a v e p r e v i o u s l y d e s c r i b e d a t i m ea v e r a g e d m a s s s p e c t r o s c o p i c m e t h o d w i t ha n a d e q u a t e l i m i t o f d e t e c t i o n ( 4 ) . H o w e v e r ,f u l l s e n s i t i v i t y c o u l d n o t b e r e a l i z e d i n m o s ts a m p l e t y p e s b e c a u s e o f in t e r f e r e nc e f r o mDDE ( a m a j o r d e g r a d a t i o n p r o d u c t o fDDT) a n d p o l y c h l o r i n a t e d b i p h e n y l s( P C B s ) . I n t h i s p a p e r we d e s c r i b e a c l e a n -u p p r o c e d u r e t h a t o v e r c o m e s t h i s d i f f i c u l t y .H o m o g e n i z e d s a m p l e s a r e s a p o n i f i e d i n a l -c o h o l i c p o t a s s i u m h y d r ox i d e a n d e x t r a c t e dw i t h h e x a n e . T h e e x t r a c t i s s h a k e n w i t h s u l -f u r i c a c i d a n d c h r o m a t o g r a p h e d o n a l u m i n a .E l u t i o n w i t h c a r b o n t e t r a c h l o r i d e - h e x a n er e m o v e s m o s t o f t h e DDE a n d P C B s . C h l o r i -n a t e d d i o x i n s a r e t h e n e l u t e d w i t h d i c h l o r o -m e t h a n e - h e x a n e . T h e TCDD c o n t a i n i n g f r a c -t i o n i s f u r t h e r p u r i f i e d b y p r e p a r a t i v e g a s -l i q u i d c h r o m a t o g r a p h y a n d a n a l y z e d b y m a s ss p e c t r o s c o p y b y u s e o f a m u l t i c h a n n e l a n a l y -z e r t o a v e r a g e s u c c e s s i v e s c a n s .We a l s o r e p o r t t h e l e v e l s o f TCDD f o u n di n a l i m i t e d n u m b e r o f s a m p l e s o f f i s h a n dc r u s t a c e a n s f r o m l o c a t i o n s i n S o u t h V i e t n a mn e a r a r e a s h e a v i l y e x p o s e d t o 2 , 4 , 5 - T .

S e p t e m b e r 1 9 7 3 2 7


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H e x a n e ( p e s t i c i d e g r a d e , F i s h e r S c i e n t i -f i c ) , d i c h l o r o m e t h a n e ( r e a g e n t g r a d e , E a s t -m a n ) , c a r b o n t e t r a c h l o r i d e ( r e a g e n t g r a d e ,M e r c k ) , 9 5 - 9 7 % s u l f u r i c a c i d ( r e a g e n tg r a d e , D u p o n t ) , s o d i u m c a r b o n a t e ( p o w -d e r e d ) ( r e a g e n t g r a d e , M a l l in c k r o d t ) , a n de t h a n o l ( p e s t i c i d e g r a d e , M a t h e s o n , C o l e m a na n d B e l l ) were u s e d .A c t i v a t e d a l u m i n a was F i s h e r A - 5 4 0 ,a c t i v a t e d a t 1 3 0 0 C f o r 2 4 h r .T h e ga s c h r o m a t o g r a p h was a B e n d i x Mo-d e l 2 2 0 0 e q u i p p e d w i t h a t h e r m a l c o n d u c t i v -i t y d e t e c t o r . T h e c o l u m n was 5 % o S E - 3 0 o n6 0 / 8 0 C h r o m o s o r b W, 2 m x 2 mm ( i d )s t a i n l e s s s t e e l . T h e t r a p f o r p r e p a r a t i v e gasc h r o m a t o g r a p h y was a 1 5 0 mm x 1 . 5 mm( i d ) g l a s s t u b e p a c k e d w i t h 3 0 mm o f g l a s sw o o l .An A s s o c i a t e d E l e c t r i c a l I n d u s t r i e s MS-9d o u b l e f o c u s i n g mass s p e c t r o m e t e r a n d aV a r i a n 1 0 2 4 t i m e - a v e r a g i n g c o m p u t e r i n t e r -f a c e d w i t h t h e MS-9 as d e s c r i b e d e a r l i e r( 4 ) , were u s e d .C l e a n u p P r o c e d u r e f o r t h e A n a l y s i s o fTCDD i n T i s s u e S a m p l e s

( 1 ) T h e s a m p l e was w e i g h e d a n d h o m o -g e n i z e d w i t h 1 . 0 - 1 . 2 p a r t s E t O H .( 2 ) T h i s h o m o g e n a t e was t r a n s f e r r e d t oa r o u n d - b o t t o m e d f l a s k e q u i p p e d w i t h a re-f l u x c o n d e n s e r ( T e f l o n t a p e u s e d o n t h eg r o u n d g l a s s j o i n t ) . T h e s a m p l e was s p i k e dw i t h a p p r o x i m a t e l y 1 0 0 0 p p t 3 7 C l TCDD; 2p a r t s 4 0 % o aqueous KOH were a d d e d , a n dt h i s m i x t u r e was r e f l u x e d f o r 2 h r . O n ep a r t a l w a y s r e f e r s t o t h e o r i g i n a l s a m p l e s .( 3 ) T h e s o l u t i o n was p a r t i a l l y c o o l e d a n d1 p a r t h e x a n e a d d e d .

( 4 ) T h e s o l u t i o n was t r a n s f e r r e d t o a sep-a r a t o r y f u n n e l , a n d t h e p h a s e s were sepa-r a t e d . T h e aqueous p h a s e was e x t r a c t e d w i t ht h r e e more i d e n t i c a l p o r t i o n s o f h e x a n e ; t h eh e x a n e e x t r a c t s were c o m b i n e d a n d c o l l e c t e di n t h e o r i g i n a l r o u n d - b o t t o m e d f l a s k .( 5 ) Th e h e x a n e p h a s e was t r a n s f e r r e d t ot h e s e p a r a t o r y f u n n e l , t h e r o u n d - b o t t o m e df l a s k was r i n s e d t w i c e w i t h a f e w m i l l i l i t e r s

o f EtOH a n d t h e n t w i c e w i t h a f e w m i l l i l i t e r so f h e x a n e ; t h e s o l v e n t w a s r e f l u x e d e a c ht i m e ; a n d t h e h e x a n e w a s e x t r a c t e d w i t h 1p a r t 1 . O N NaOH.( 6 ) T h e h e x a n e w a s e x t r a c t e d f o u r t i m e s( o r u n t i l a c i d p h a s e w a s c o l o r l e s s ) w i t h 2p a r t s 9 5 - 9 7 % o H 2 S O 4 . E m u l s i o n s w e r e b r o k e nw i t h a f e w d r o p s o f s a t u r a t e d N a 2 C O 3s o l u t i o n .( 7 ) T h e h e x a n e w a s e x t r a c t e d w i t h 1 p a r tw a t e r , a n d s e v e r a l g r a m s o f N a 2 0 C 3 w e r ea d d e d t o t h e h e x a n e .( 8 ) T h e h e x a n e w a s f i l t e r e d t h r o u g h ac o l u m n o f N a 2 C O , ( 1 0 0 mm x 1 0 mm i d f o r3 0 0 m l h e x a n e ) , t h e N a 2 C O 3 f i r s t b e i n g p r e -w a s h e d w i t h s e v e r a l m i l l i l i t e r s o f h e x a n e .( 9 ) T h e h e x a n e w a s c o n c e n t r a t e d t o 3 - 4m l ( S n y d e r c o l u m n ) .

( 1 0 ) T h e h e x a n e r e s i d u e w a s c h r o m a t o -g r a p h e d o n a c o l u m n o f a c t i v a t e d A 2 0 3 ( 5 0mm i n a 5 mm d i s p o s a b l e p i p e t ) . T h e c o l u m ns h o u l d n o t b e p r e w a s h e d . E l u t i o n was w i t h 1 2m l o f 2 0 % o C C G 4 i n h e x a n e , t h e n 1 m l o f h e x a -a n e , a n d f i n a l l y 4 m l o f 2 0 % o C H 2 C 1 2 i nh e x a n e .( 1 1 ) T h e 2 0 % o C H 2 C l 2 f r a c t i o n was c o n -c e n t r a t e d c a r e f u l l y t o a b o u t 5 0 F d , 1 0 0 - 2 0 0, u l b e n z e n e a d d e d , a n d c o n c e n t r a t i o n r e -p e a t e d t o 2 0 F J .( 1 2 ) A f e w m i c r o g r a m s o f m - t e r p h e n y li n b e n z e n e w e r e a d d e d t o t h e r e s i d u e a n dt h e m i x t u r e s u b j e c t e d t o p r e p a r a t i v e c h r o -m a t o g r a p h y . T h e r e t e n t i o n t i m e o f m - t e r -p h e n y l r e l a t i v e t o t h a t o f TCDD was d e t e r -m i n e d b e f o r e h a n d a n d u s e d t o m a k e c e r t a i n

t h a t t h e TCDD c o l l e c t i o n was c a r r i e d o u t a tt h e r i g h t r e t e n t i o n t i m e .( 1 3 ) T h e GLC t r a p c o n t a i n i n g TCDDw a s e l u t e d w i t h 6 0 , u f o l l o w e d b y 1 0 , u l o fb e n z e n e . T h e t o t a l a m o u n t o f e l u a n t c o l l e c t e dw a s m e a s u r e d , a n d t h e f r a c t i o n s i z e f o r t h ep l a n n e d n u m b e r o f f r a c t i o n s ( t y p i c a l l y t e n )c a l c u l a t e d .( 1 4 ) T h e f r a c t i o n s f o r TCDD a n a l y s i sw e r e p r e p a r e d i n t h e s a m p l e t u b e s d e s c r i b e dp r e v i o u s l y ( 4 ) . A known amount of TCDDw a s a d d e d t o t h r e e o r more f r a c t i o n s f o rq u a n t i t a t i o n o f a n y TCDD o b s e r v e d . T h ea m o u n t o f TCDD a d d e d p e r f r a c t i o n f o r

E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s8


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q u a n t i t a t i o n s h o u l d b e a p p r o x i m a t e l y t h r e eo r f ou r t im e s t h e a m o u n t e x p e c t e d t o b ep r e s e n t .( 1 5 ) T h e f r a c t i o n s w e r e a n a l y z e d w i t h t h eMS-9 i n s t r u m e n t . T y p i c a l c o n d i t i o n s w e r e :s o u r c e 2 2 0 C , r e s o l u t i o n 1 0 , 0 0 0 ( b a s e do n a 10% v a l l e y b e t w e e n p e a k s ) , t r a p c u r -r e n t 1 . 0 mA ( r h e n i u m f i l a m e n t ) , e l e c t r o nm u l t i p l i e r 7 0 0 , i o n i z i n g v o l t a g e 7 0 e V , t i m ea v e r a g i n g a t f o u r s c a ns p e r s e c o n d .( 1 6 ) P e a k h e i g h t s w e r e m e a s u r e d a t m/e3 2 1 . 8 9 4 . T h e q u a n t i t y o f TCDD ( p i c a g r a m s ) ,p r e s e n t i n t h e f r a c t i o n s t o w h i c h TCDDh a s n o t b e e n a d d e d w a s c o m p u t e d f r o m t h er a t i o o f t h e i r mean p e a k h e i g h t s t o t h e m e a np e a k h e i g h t s f o u n d w i t h a d d e d TCDD.( 1 7 ) S t e p s ( 1 4 ) - ( 1 6 ) w e r e r e p e a t e d , b u t3 7 C 1 TCDD w a s a d d e d a n d p e a k h e i g h t s w e r em e a s u r e d a t m/e 3 2 7 . 8 8 5 i n o r d e r t o c o m p u t et h e a m o u n t o f 3 7 C l TCDD r e c o v e r e d . T h er e c o v e r y t h r o u g h t h e c o m p l e t e c l e a nu p p r o -c e d u r e w a s t h e n c a l c u l a t e d b a s e d o n t h ea m o u n t o f 3 7 C l TCDD a d d e d t o t h e s a m p l ea t t h e b e g i n n i n g o f t h e c l e a n u p .( 1 8 ) T h e q u a n t i t y o f TCDD c o m p u t e di n s t e p ( 1 5 ) w a s c o r r e c t e d b y t h e r e c o v e r y

f a c t o r o b t a i n e d i n s t e p ( 1 6 ) t o g i v e t h ef i n a l r e s u l t .S a m p l e C o l l e c t i o n

F r e s h l y c a u g h t f i s h a n d c r u s t a c e a n s w e r ec o l l e c t e d i n S o u t h V i e t n a m i n A u g u s t a n dS e p t e m b e r 1 9 7 0 f r o m l o c a l f i s h e r m e n . T h es a m p l e s w e r e h o m o g e n i z e d w i t h a m e a tg r i n d e r , p l a c e d i n a c e t o n e - r i n s e d g l a s s b o t -t l e s w i t h a l u m i n u m f o i l - l i n e d c a p s , a n d i m -m e d i a t e l y f r o z e n i n s o l i d C O 2 . L a t e r o n t h es a m e d a y , s a m p l e s w e r e p l a c e d i n a L i n d eL R - 3 5 l i q u i d n i t r o g e n r e f r i g e r a t o r w h e r et h e y r e m a i n e d u n t i l a n a l y s i s . W a t e r b l a n k sw e r e p r e s e n t i n t h e l i q u i d n i t r o g e n r e f r i g e r a -t o r t h r o u g h o u t t h e s t o r a g e p e r i o d a n d w e r ea n a l y z e d w i t h t h e s a m p l e s . F r e s h C a p e C o db u t t e r f i s h ( P o r o n o t u s t r i c a n t h u s , f a m i l yS t r o m a t e i d a e ) w e r e o b t a i n e d f r o m a l o c a lm a r k e t , h o m o g e n i z e d , a n d k e p t a t - 2 0 Cu n t i l a n a l y s i s . D o m e s t i c b e e f l i v e r s w e r e o b -t a i n e d a n d t r e a t e d s i m i l a r l y .

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Th e mass s p e c t r a o f n a t u r a l a n d 3 7 C ITCDD ar e s h o w n i n F i g u r e 1 . T h e m o s ti n t e n s e s ig na l f o r n a t u r a l TCDD occurs a tm/e 3 2 1 . 8 9 4 ( n o m i n a l m/e 3 2 2 ) , c o r r e s p o n d -i n g t o t h e i s o t o p i c i s o m e r w i t h one atomo f 3 7 C l a n d t h r e e a t o m s o f 3 - 5 C . T h e n a t u r a la b u n d a n c e s o f t h e C l i s o t o p e s are 7 5 . 5 3 a n d2 4 . 4 7 % o , r e s p e c t i v e l y . T h e o b s e r v e d s p e c t r u mf o r t h e s y n t h e t i c 3 7 C I TCDD c o r r e s p o n d s t oan i s o t o p i c p u r i t y o f 9 5 . 5 % o 3 7 C I , t h e same ast h e v a l u e c l a i m e d b y t h e m a n u f a c t u r e r ( O a kR i d g e N a t i o n a l L a b o r a t o r y ) o f t h e N a C l u s e di n t h e s y n t h e s i s o f t h e l a b e l e d TCDD. Th es y n t h e t i c 3 7 C l TCDD c o n t r i b u t e s o n l y 0 . 0 4 2 % oas much t o t h e p e a k a t m/e 3 2 2 as i t c o n t r i b -u t e s t o i t s m o s t i n t e n s e s i g n a l a t m/e 3 2 8 .T h e c o n t r i b u t i o n a t m/e 3 2 0 i s even l o w e r ,b y a f a c t o r o f n e a r l y 1 0 0 . T h i s a l l o w san excess o f 3 7 C l TCDD t o b e a d d e d t o e a c hs a m p l e b e f o r e c l e a n u p w i t h o u t i n t e r f e r i n g

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F I G U R E 1 . M a s s s p e c t r a o f ( A ) TCDD a n d ( B )3 7 C l - l a b e l e d TCDD. T h e i s o t o p i c p u r i t y o f t h e7 C l i s 9 5 . 5 % . T h e a s t e r i s k d e n o t e s an i m p u r i t y .T h e m u l t i p l i c i t y o f l i n e s a s s o c i a t e d w i t h e a c h m a-j o r m o l e c u l a r s p e c i e s r e s u l t s from t h e p r e s e n c eo f v a r i o u s i s o t o p e s o f C 1 a n d C .

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3 2 1 . 9 0 0 3 2 .M / E

P T n , T R . 9 2 M o n . n i p + . r a ohninD r l i l i t - n i n

m a k e s p o s s i b l e t h e m e a s u r e m e n t o f p i c o -gram q u a n t i t i e s o f TCDD i n s a m p l e s i n i t i a l l yc o n t a i n i n g m o r e t h a n a m i l l i o n f o l d e x c e s so f DDE a n d P C B s . F i g u r e 2 s h o w s t h e e f -f e c t i v e n e s s o f t h i s p r o c e d u r e .T h e c a l c u l a t i o n o f TCDD l e v e l s d e s c r i b e di n s t e p s ( 1 4 ) - ( 1 6 ) o f t h e e x p e r i m e n t a l s e c -t i o n a s s u m e s a l i n e a r r e l a t i o n s h i p b e t w e e np e a k h e i g h t a n d a m o u n t o f TCDD p r e s e n ti n a n y g i v e n s a m p l e . F i g u r e 3 d e m o n s t r a t e st h a t t h e r e s p o n s e i s i n d e e d l i n e a r o v e r t h ef u l l r a n g e o f TCDD a m o u n t s i n t r o d u c e d i n t ot h e MS-9 i n t h e c o u r s e o f t h e a n a l y s e s r e -

B . p o r t e d h e r e .T h e r e p r o d u c i b i l i t y a n d o v e r a l l r e c o v e r yo f t h e c o m p l e t e a n a l y t i c a l p r o c e d u r e i s i l l u -s t r a t e d i n T a b l e 1 . A s a m p l e o f b e e f l i v e r w a sh o m o g e n i z e d a n d d i v i d e d i n t o t h r e e p o r t i o n se a c h o f w h i c h w a s t h e n s p i k e d w i t h 2 0 p p tTCDD a n d 1 0 0 0 p p t 3 7 C l TCDD. T h e t h r e es a m p l e s w e r e i n d e p e n d e n t l y p u t t h r o u g h t h e

A . c l e a n u p p r o c e d u r e up t o t h e GLC s t e p . E a c hs a m p l e w a s t h e n s p l i t i n t o t h r e e p o r t i o n s b e -f o r e p r e p a r a t i v e CLC a n d m a s s s p e c t r o m e t -r i c a n a l y s i s , g i v i n g r i s e t o a t o t a l o f n i n es e p a r a t e v a l u e s f o r t h e r e c o v e r y o f b o t h'- I TCDD a n d 3 7 C l T C D D . T h e a v e r a g e r e -1 . 8 0 0 c o v e r y w a s 3 4 7% f o r TCDD a n d 2 7 5% f o r 3 7 C l TCDD. When t h e s l i g h t b a c k -n f n n r . g r o u n d s i g n a l a t m / e 3 2 2 i n a n u n s p i k e dr Xl:UM Q . ITIUMSZSPVXVrU 151UWllls rrU u> U %;WU 1VI""

a n d P CB l e v e l s i n f i s h r e s i d u e by means o f a l u m i n ac h r o m a t o g r a p h y . F o l l o w i n g t h e s u l f u r i c a c i dc l e a n u p s t e p , t h e r e s i d u e i n h e x a n e i s a d d e d t o ac o l u m n o f a c t i v a t e d a l u m i n a : ( A ) T r a c e from t h em a t e r i a l e l u t e d b y 20 % C H 2 C 1 2 i n h e x a n e a f t e r t h ec o l u m n was f i r s t e l u t e d w i t h 20 % C C l . i n h e x a n e ;( B ) t r a c e o b t a i n e d from a s i m i l a r 20% C H 2 C l 2 -i n - h e x a n e e l u t i o n a f t e r t h e c o l u m n was f i r s t e l u t e dw i t h 1% C H 2 C l 2 i n h e x a n e . E l u t i o n w i t h 1 %C H 2 0 1 2 i n h e x a n e was r e p o r t e d t o b e e f f e c t i v e i nr e d u c i n g t h e amount o f P CB r e s i d u e s ( 5 ) . E l u t i o nw i t h 20 % C C L 4 i s c l e a r l y even more e f f e c t i v e a n dwas r o u t i n e l y u s e d i n o b t a i n i n g t h e r e s u l t s re-p o r t e d h e r e .

w i t h a n a l y s i s o f n a t u r a l TCDD a t m / e 3 2 2a n d 3 2 0 . T h e a d d i t i o n o f 3 7 C l TCDD p r o v i d e sa c a r r i e r a n d m a k e s p o s s i b l e t h e c a l c u l a -t i o n o f a b s o l u t e r e c o v e r i e s .An a l u m i n a c h r o m a t o g r a p h y s t e p h a s b e e nd e v e l o p e d w h i c h , when c o m b i n e d w i t h t h ec l e a n u p s t e p s d e s c r i b e d p r e v i o u s l y , ( 4 )

6 0 8 0TCDD ( p g )

F I G U R E 3 . L i n e a r i t y o f response f o r TCDD i n t h epresence o f b e e f l i v e r r e s i d u e . Th e TCDD v a l u e sare t h e a m o u n t s i n t r o d u c e d i n t o i n d i v i d u a l r u n son t h e M S - 9 .

E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s

3 2 2 . 0 0 0

3 0


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T a b l e 1 . R e c o v e r i e s o f TCDD ( a d d e d a t 2 0 p p t ) a n d' 7 C l TCDD ( a d d e d a t 1 0 0 0 p p t ) f r o m b e e f l i v e r .R e c o v e r y , %

S a m p l e TCDD ' 7 C l TCDDS a m p l e AGLC 1 4 7 2 4GLC 2 3 6 3 0GLC 3 3 6 2 5S a m p l e BGLC 1 2 8 3 5GLC 2 3 1 2 9GLC 3 2 4 2 0S a m p l e CGLC 1 2 9 2 7GLC 2 4 0 3 2GLC 3 3 7 2 1Mean r e c o v e r y 3 4 7 . 2 2 7 5 . 0

f o r A , B , a n dC

s a m p l e o f t h e same l i v e r i s t a k e n i n t o ac -c o u n t , t h e c a l c u l a t e d r e c o v e r i e s f r o m t h es p i k e d s a m p l e s b e c o m e even more n e a r l ye q u a l . E x p e r i m e n t s p e r f o r m e d s e p a r a t e l yw i t h e a c h i n d i v i d u a l c l e a n u p s t e p e s t a b l i s h e dt h a t t h e s t e p w i t h l o w e s t recovery i s prepara-t i v e g a s - l i q u i d c h r o m a t o g r a p h y .We c o n c l u d e from t h e s e and o t h e r c o n t r o l st h a t t h e p r e s e n t a n a l y t i c a l m e t h o d pro-v i d e s t h e s e n s i t i v i t y a n d r e p r o d u c i b i l i t y re-q u i r e d f o r b i o l o g i c a l l y m e a n i n g f u l a n a l y s e so f a n i m a l t i s s u e s a m p l e s . T h e m e t h o d m a k e sp o s s i b l e i n v e s t i g a t i o n s o f s u c h , s a m p l e s a tl e v e l s a p p r o x i m a t e l y 1 0 - 4 t i m e s t h o s e r e p o r t -e d h e r e t o f o r e ( 6 ) .

O b s e r v e d TCDD L e v e l sS i g n a l s a t m / e 3 2 0 a n d 3 2 2 were con-s p i c u o u s l y p r e s e n t i n e a c h o f t h e f i s h a n dc r u s t a c e a n s a m p l e s f r o m V i e t n a m . T h e c a l -c u l a t e d l e v e l s o f TCDD, s u m m a r i z e d i n T a b l e2 , range from 1 8 p p t t o 8 1 4 p p t , b a s e d ont o t a l w e t b o d y w e i g h t .No p e a k was o b s e r v e d a t m / e 3 2 0 or 3 2 2w i t h C a p e C o d b u t t e r f i s h . T h e b a c k g r o u n ds i g n a l c o r r e s p o n d e d t o a l e v e l o f 3 p p t o fTCDD. No p e a k s were o b s e r v e d i n w a t e rb l a n k s a m p l e s p r e s e n t i n t h e l i q u i d n i t r o g e nr e f r i g e r a t o r t h r o u g h o u t t h e s a m p l e c o l l e c -t i o n a n d s t o r a g e p e r i o d .C o n f i r m a t i o n t h a t p e a k s o b s e r v e d a t m / e3 2 0 a n d 3 2 2 are i n f a c t p r o d u c e d b y TCDDi s r o u t i n e l y p r o v i d e d b y t h e c r i t e r i a o u t -l i n e d i n p a r t A o f T a b l e 3 . A l l t h r e e o f t h e s ec r i t e r i a are m e t b y t h e mass s p e c t r a f r o me a c h o f t h e V i e t n a m e s e s a m p l e s .T h e a d d i t i o n a l c o n f i r m a t o r y p r o c e d u r e sl i s t e d i n p a r t B o f T a b l e 3 were c a r r i e d o u t

o n a s a m p l e o f V i e t n a m e s e f i s h . T h i s s a m p l e ,carp f r o m t h e Dong N a i R i v e r , e x h i b i t e d amean TCDD l e v e l o f 5 4 0 p p t . T h e masss p e c t r u m i n t h e r e g i o n m / e 3 2 2 i s s h o w ni n F i g u r e 4 . T h e compound o b s e r v e d i nt h i s f i s h b e h a v e d i d e n t i c a l l y t o TCDD i n e a c ho f t h e t h r e e a d d i t i o n a l c o n f i r m a t o r y t e s t s .We c o n s i d e r i t e x t r a o r d i n a r i l y u n l i k e l y t h a tt h i s - c o m p o u n d i s a n y t h i n g o t h e r t h a n a t e t -r a c h l o r o d i b e n z o - p - d i o x i n . I n c o n t r a s t t o t h es i g n i f i c a n t a m o u n t s o f 2 , 3 , 7 , 8 - t e t r a c h l o r o d i -

T a b l e 2 . TCDD l e v e l s i n f i s h a n d c r u s t a c e a n s .L e v e l , p p t t o t a l w e t b o d yw e i g h t bM ap s i t e a I I I I I I Mean

A D o n g N a i R i v e r ( i n t e r i o r ) C a r p ( C y p r i n i n a e ) 6 9 0 3 2 0 6 1 0 5 4 0B Dong N a i R i v e r ( i n t e r i o r ) C a t f i s h ( S i l u r i d a e ) 6 1 0 1 0 2 0 8 1 0B Dong N a i R i v e r ( i n t e r i o r ) C a t f i s h ( T a c h y s u r i d a e ) 5 1 0 5 3 0 5 2 0C S a i Go n R i v e r ( i n t e r i o r ) C a t f i s h ( S c h i l b e i d a e ) 5 2 8 9 7 0C S a i Go n R i v e r ( i n t e r i o r ) R i v e r Prawn ( P a l a e m o n i d a e ) 3 4 4 9 4 2D Can G i o V i l l a g e ( s e a c o a s t ) C r o a k e r ( S c i a e n i d a e ) 1 1 0 4 9 7 9D C a p e G i o V i l l a g e ( s e a c o a s t ) P r a w n ( P e n e i d a e ) 2 3 1 4 1 8C a p e C o d , M a s s a c h u s e t t s B u t t e r f i s h ( S t r o m a t e i d a e )


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T a b l e 3 . C o n f i r m a t i o n P r o c e d u r e sA . R o u t i n e1 . F o l l o w s ' 7 C l TCDD t h r o u g h h i g h l y s p e c i f i cc l e a n u p2 . Has e x p e c t e d m a s s ( 2 - 3 m m u ) a t m/e 3 2 0a n d 3 2 23 . Has e x p e c t e d r a t i o o f i s o t o p i c i s o m e r s a tm / e 3 2 0 a n d 3 2 2B . A d d i t i o n a l '1 . M + - C O C l f r a g m e n t a t i o n p e a k h a s e x p e c t e dm a s s a n d i s o t o p i c i s o m e r r a t i o2 . P e r c e n t r e c o v e r y a f t e r p a r t i a l p h o t o l y t i c d e -c o m p o s i t i o n e q u a l s t h a t o f ' 7 C l TCDD ( 7 , 8 )3 . P a r t i t i o n c o e f f i c i e n t b e t w e e n d i c h l o r o m e t h a n e -h e x a n e a n d a c e t o n i t r i l e e q u a l s t h a t o f 5 C lTCDD ( 7 ) .

a S t e p s 2 a n d 3 o f t h e a d d i t i o n a l p r o c e d u r e sw e r e c a r r i e d o u t o n t h e d i c h l o r o m e t h a n e - h e x a n ee l u a n t f r o m t h e a l u m i n a c h r o m a t o g r a p h y p r i o rt o p r e p a r a t i v e G L C .

A . V I E T N A M E S E C A R P P L U S T C D D

B . V I E T N A M E S E C A R P

F I G U R E 5 . M ap s h o w i ng s a m p l i ng s i t e s i n r e l a t i o n t or i v e r s a n d p r i n c i p a l s p r a y e d a r e a s . S i t e s A a n dB a r e l oc at e d o n t h e Dong N a i R i v e r , s i t e C i so n t h e S a i Go n R i v e r , a n d s i t e D i s o n t h e c o a s ta t Can G i o . S p r a y e d a r e a s a r e d e p i c t e d o n l yw i t h i n t h e r e g i o n b o u n d e d b y t h e d a s h e d l i n e s

A p r i l 1 9 7 0 a r e s h o w n a s s t i p l e d . T h e n u m b e ro f s a m p l e s i s n o t a d e q u a t e t o p e r m i t r e l i a b l ec o n c l u s i o n s c o n c e r n i n g t h e d i f f e r e n c e s b e -t w e e n v a r i o u s l o c a t i o n s a n d s p e c i e s , a l t h o u g ht h i s c e r t a i n l y s h o u l d b e a s u b j e c t o f f u t u r es t u d i e s .C . C A P E C O D B U T T E R F I S H

3 2 2 . 0 0 0 3 2 1 . 9 0 0 3 2 1 . 8 0 0F I G U R E 4 . TCDD s i g n a l s o b s e r v e d i n f i s h s a m p l e s :( A ) V i e t n a m e s e c a r p p l u s 6 0 p g TCDD, ( w e tw e i g h t o f f i s h 0 . 1 8 g ) ; ( B ) V i e t n a m e s e c a r p , ( w e tw e i g h t o f f i s h 0 . 1 8 g ) ; ( C ) C a p e C o d b u t t e r f i s h( w e t w e i g h t o f f i s h 0 . 1 6 g ) .b e n z o - p - d i o x i n known t o h a v e b e e n d i s s e m i -n a t e d a s a c o n t a m i n a n t o f 2 , 4 , 5 - T ( 1 ) . , weknow o f n o l i k e l y r o u t e b y w h i c h o t h e r i s om -e r s o f TCDD m i g h t h a v e b e e n i n t r o d u c e d i n t ot h e V i e t n a m e s e e n v i r o n m e n t .T h e l o c a t i o n s f r o m w h i c h t h e V i e t n a m e s es a m p l e s w e r e o b t a i n e d a r e d e s i g n a t e d i n F i g -u r e 5 . T h e l e t t e r s c o r r e s p o n d t o t h o s e i nT a b l e 2 . A r e a s h e a v i l y t r e a t e d w i t h 2 , 4 , 5 - Tb e f o r e i t s u s e w a s o r d e r e d d i s c o n t i n u e d i n

D i s c u s s i o nC o n s i d e r i n g t h e l i m i t e d n u m b e r o f s a m -p l e s we h a v e a n a l y z e d a n d t h e f a c t t h a t t h e yw e r e c o l l e c t e d 2 1 / 2 y r a g o , i t d o e s n o t s e e ma p p r o p r i a t e t o a t t e m p t a n y d e t a i l e d e v a l u a -t i o n o f t h e p o s s i b l e t o x i c o l o g i c a l s i g n i f i c a n c eo f o u r r e s u l t s . S u c h d i s c u s s i o n i s made e v e nm o r e d i f f i c u l t b y t h e c o m p l e x i t y a n d i n -c o m p l e t e n e s s o f t h e e x i s t i n g t o x i c o l o g i c a ld a t a . H o w e v e r , i n o r d e r t o p r o v i d e p e r s p e c -t i v e f o r s u c h d i s c u s s i o n , a t a b u l a t i o n o f s o m eo f t h e p r i n c i p a l t o x i c i t y d a t a o n TCDD i sp r e s e n t e d i n T a b l e 4 . I t may b e n o t e d t h a t

g u i n e a p i g s c o n s u m i n g t h e i r w e i g h t o f f o o dc o n t a m i n a t e d w i t h TCDD a t a l e v e l o f 6 0 0p p t w o u l d h a v e i n g e s t e d a q u a n t i t y c o r -r e s p o n d i n g t o t h e l e t h a l d o s e . I n c o n t r a s t ,a f a r g r e a t e r q u a n t i t y o f TCDD i s r e q u i r e dt o r e a c h t h e L D 5 0 c i t e d f o r r a t s . T h e t a b l es h o w s t h a t t e r a t o g e n e s i s i n t h e r a t o c c u r sa t d o s e s s u b s t a n t i a l l y l o w e r t h a n t h o s e r e -q u i r e d t o k i l l .E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s2


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T a b l e 4 . L e v e l s o f TCDD g i v i n g v a r i o u s b i o l o g i c a l e f f e c t s .TCDD t o o b t a i n e f f e c t ,E f f e c t p p t b o d y w e i g h t R e f e r e n c e

L e t h a l i t yF e m a l e r a t , s i n g l e o r a l d o s e L D 5 o( o b s e r v a t i o n s t e r m i n a t e d a t 4 4 d a y s ) 4 5 , 0 0 0 ( 8 )M a l e r a t , s i n g l e o r a l d o s e L D 5 o( o b s e r v a t i o n s t e r m i n a t e d a t 4 4 d a y s ) 2 3 , 0 0 0 ( 8 )M a l e g u i n e a p i g , s i n g l e o r a l d o s e L D 5 o( o b s e r v a t i o n s t e r m i n a t e d a t 5 0 d a y s ) 6 0 0 ( 8 )T e r a t o g e n i c i t yC l e f t p a l a t e i n 5 0 % o N M R I m i c e , d a i l y o r a l d o s e ,d a y s 6 - 1 5 5 , 0 0 0 ( 9 )I n t e s t i n a l h e m o r r h a g e a n d s u b c u t a n e o u s e d e m a i n 5 0 % , 1 2 5 - 5 0 0 ( 3 )S p r a g u e - D a w l e y r a t s , d a i l y o r a l d o s e , d a y s 6 - 1 5Edema a n d d e a t h i n c h i c k e n e m b r y o , s i n g l e i n j e c t i o n . 2 0 ( 1 0 )Enzyme i n d u c t i o nD o u b l i n g o f 8 - a m i n o l e v u l i n i c a c i d s y n t h e t a s e i nc h i c k e n e m b r y o , s i n g l e i n j e c t i o n 3 0 ( 1 1 )

M i t o t i c a r r e s tL i l y e n d o s p e r m , a m b i e n t c o n c e n t r a t i o n


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f o r p o s s i b l e c o n n e c t i o n s w i t h h e r b i c i d e e x -p o s u r e ( 1 4 ) . I t i s o f o b v i o u s i n t e r e s t t o s u r -v e y a p p r o p r i a t e l y c h o s e n p o p u l a t i o n s i nS o u t h V i e t n a m m o r e c l o s e l y , e s p e c i a l l y i fTCDD r e s i d u e s s h o u l d b e f o u n d i n humant i s s u e s a m p l e s .F i n a l l y , t u r n i n g f r o m q u e s t i o n s o f e n v i r o n -m e n t a l t o x i c o l o g y t o t h e b i o l o g i c a l m e c h a n -i s m s o f a c t i o n , we n o t e t h a t TCDD s e e m s t ob e p a r t i c u l a r l y t o x i c t o p r o l i f e r a t i n g t i s s u e s ,a s s u g g e s t e d b y i t s e f f e c t s o n s p e r m a t o g e n e s i sa n d h e m a t o p o i e s i s a n d i t s a p p a r e n t t o x i c i t yt o t h e i n t e s t i n a l e p i t h e l i u m ( 1 3 ) a n d t h et h y m u s ( 1 5 ) . T h e s e i n d i c a t i o n s a r e c o n s i s -t e n t w i t h t h e e f f e c t s o f a m i t o t i c p o i s o n ,s u c h a s TCDD i s known t o b e i n t h e A f r i c a nb l o o d l i l y ( 1 2 ) a n d p o s s i b l y i n D r o s o p h t l am e l a n o g a s t e r ( 1 6 ) . We a r e l e d b y t h e s e o b -s e r v a t i o n s t o s p e c u l a t e t h a t TCDD may b ea b l e c a t a l y t i c a l l y t o d i s r u p t m i c r o t u b u l e s ,t h e s u b c e l l u l a r e l e m e n t s o f w h i c h s p i n d l ef i b e r s a r e c o n s t r u c t e d a n d w h i c h a r e u b i -q u i t o u s i n t h e i r s t r u c t u r a l r o l e s i n c e l l e x -t e n s i o n a n d c e l l m o v e m e n t .S u m m a r yA p r o c e d u r e h a s b e e n d e v e l o p e d f o r t h er e l i a b l e d e t e c t i o n o f TCDD i n a n i m a l t i s s u e sdown t o l e v e l s a p p r o a c h i n g 1 p p t . I t m a k e su s e o f c h e m i c a l c l e a n u p , p r e p a r a t i v e g a s -l i q u i d c h r o m a t o g r a p h y , a n d a n a l y s i s b y t i m e -

a v e r a g e d h i g h r e s o l u t i o n m a s s s p e c t r o s c o p y .A l i m i t e d n u m b e r o f f i s h a n d c r u s t a c e a ns a m p l e s w a s c o l l e c t e d i n S o u t h V i e t n a m i n1 9 7 0 n e a r a r e a s h e a v i l y e x p o s e d t o t h e h e r b i -c i d e 2 , 4 , 5 - T . TCDD w a s d e t e c t e d i n t h e s es a m p l e s a t l e v e l s r a n g i n g f r o m 1 8 t o 8 1 0p p t . TCDD w a s n o t d e t e c t e d i n a s a m p l e o fC a p e C o d b u t t e r f i s h u s e d a s a c o n t r o l .

T h e s e r e s u l t s s u g g e s t t h a t TCDD may h a v ea c c u m u l a t e d t o b i o l o g i c a l l y s i g n i f i c a n t l e v e l si n f o o d c h a i n s i n s o m e a r e a s o f S o u t h V i e t -nam e x p o s e d t o h e r b i c i d e s p r a y i n g .N o t e a d d e d i n p r oo f : O v e r a l l r e c o v e r i e s h a v eb e e n i n c r e a s e d t o 6 0 - 8 0 % o b y r e p l a c in g t h eGLC s t e p w i t h a n a d d i t i o n a l A 1 2 0 3 c o l u m ns t e p . D e t a i l s o f t h i s p r o c e d u r e w i l l b e d e -s c r i b e d i n a f u t u r e p u b l i c a t i o n .

A c k n o w l e d g e m e n tT h i s r e s e a r c h w a s i n i t i a t e d b y t h e H e r b i -c i d e A s s e s s m e n t C o m m i s s i o n o f t h e A m e r i -

c a n A s s o c i a t i o n f o r t h e A d v a n c e m e n t o fS c i e n c e . T h e w o r k h a s b e e n s u p p o r t e d b yf u n d s f r o m t h e AAAS, t h e F o r d F o u n d a t i o n ,a n d t h e N a t i o n a l I n s t i t u t e o f E n v i r o n m e n t a lH e a l t h S c i e n c e s (NIH g r a n t n o . 1 ROI E S -0 0 8 5 1 - 0 1 T O X ) .We t h a n k P r o f e s s o r B u i T h i L a n g , M r .R o b e r t C o o k , P r o f e s s o r A r t h u r W e s t i n g , a n dD r . J o h n C o n s t a b l e f o r a i d i n g i n t h e c o l l e c -t i o n o f s a m p l e s , a n d P r o f e s s o r L a n g f o rh e l p i n g t o i d e n t i f y t h e s p e c i m e n s c o l l e c t e d .We a l s o t h a n k K e n n e t h G r o s s a n d L e s l e yN e w t o n f o r t h e i r e x p e r t a s s i s t a n c e i n t h el a D o r a t o r y .

REFERENCES1 . R e p o r t o n 2 , 4 , 5 - T . A R e p o r t o f t h e P a n e l o nH e r b i c i d e s o f t h e P r e s i d e n t ' s S c i e n c e A d v i s o r yC o m m i t t e e , E x e c u t i v e O f f i c e o f t h e P r e s i d e n t ,O f f i c e o f S c i e n c e a n d T e c h n o l o g y , March 1 9 7 1 .2 . E f f e c t s o f 2 , 4 , 5 - T o n M an a n d t h e E n v i r o n m e n t .H e a r i n g s b e f o r e t h e S u b c o m m i t t e e o n E n e r g y ,N a t u r a l R e s o u r c e s , a n d t h e E n v i r o n m e n t o f t h eC o m m i t t e e o n C o m m e r c e , U n i t e d S t a t e s S e n a t e ,

9 1 s t C o n g r e s s , A p r i l 7 a n d 1 5 , 1 9 7 0 .3 . S p a r s c h u , G . L . , D u n n , F . L . , a n d R o w e , V . K .S t u d y o f t h e T e r a t o g e n i c i t y o f 2 , 3 , 7 , 8 - T e t r a -c h l o r o d i b e n z o - p - d i o x i n i n t h e R a t . F o o d C o s m e t .T o x i c o l . 9 : 4 0 5 ( 1 9 7 1 ) .4 . B a u g h m a n , R . , a n d M e s e l s o n , M . An i m p r o v e da n a l y s i s f o r 2 , 3 , 7 , 8 - t e t r a c h l o r o d i b e n z o - p - d i o x i n .

I n : A d v a n c e s i n C h e m i s t r y S e r . 1 2 0 , E . B l a i r ,E d . , A m e r i c a n C h e m i c a l S o c i e t y , W a s h i n g t o n ,D C . , 1 9 7 3 , p . 9 2 .5 . P o r t e r , M . L . , a n d B u r k e , J . A . S e p a r a t i o n o f

t h r e e c h l o r o d i b e n z o - p - d i o x i n s from s o m e p o l y -c h l o r i n a t e d b i p h e n y l s b y c h r o m a t o g r a p h y o n a na l u m i n u m o x i d e c o l u m n , 1 9 7 1 . J . A s s o c . O f f i c .A n a l . C h e m . 5 4 : 1 4 2 6 ( 1 9 7 1 ) .

6 . W o o l s o n , E . A . , R e i c h e l , W. I . , a n d Y o u n g , A . L .D i o x i n r e s i d u e s i n l a k e l a n d s a n d a n d e a g l es a m p l e s . I n A d v a n c e s i n C h e m i s t r y S e r . 1 2 0 ,E . B l a i r , E d . , A m e r i c a n C h e m i c a l S o c i e t y ,W a s h i n g t o n , D . C . , i n p r e s s .7 . W o o l s o n , E . A . , T h o m a s , R . F . , a n d E n s o r , P .D . J . S u r v e y o f p o l y c h l o r o d i b e n z o - p - d i o x i n c o n -

t e n t i n s e l e c t e d p e s t i c i d e s . J . A g r . F o o d C h e m .2 0 : 3 5 1 ( 1 9 7 2 ) .

8 . C r o s b y , D . G . , W o n g , A . S . , P l i m m e r , J . R . , a n d3 4 E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s


27/27

W o o l s o n , E . A . P h o t o d e c o m p o s i t i o n o f c h l o r i -n a t e d d i b e n z o - p - d i o x i n s . S c i e n c e 1 7 3 : 7 4 8 ( 1 9 7 1 ) .9 . N e u b e r t , D . , a n d D i l l m a n , I . E m b r y o t o x i c e f f e c t si n m i c e t r e a t e d w i t h 2 , 4 , 5 - T a n d t e t r a c h l o r o -d i b e n z o - p - d i o x i n . N a u n y n - S c h m i e d b e r g s A r c h .P h a r m a c o l . 2 7 2 : 2 4 3 ( 1 9 7 2 ) .

1 0 . V e r r e t , J . I n : E f f e c t s o f 2 , 4 , 5 - T o n M an a n dt h e E n v i r o n m e n t . H e a r i n g s b e f o r e t h e S u b -c o m m i t t e e o n E n e r g y , N a t u r a l R e s o u r c e s , a n dt h e E n v i r o n m e n t o f t h e C o m m i t t e e o n Com-m e r c e , U n i t e d S t a t e s S e n a t e , 9 1 s t C o n g r e s s ,A p r i l 7 a n d 1 5 , 1 9 7 0 .

1 1 . P o l a n d , A . , a n d G l o v e r , E . 2 , 3 , 7 , 8 - T e t r a c h l o r o -d i b e n z o - p - d i o x i n : a p o t e n t i n d u c e r o f 8 - a m i n o -l e v u l i n i c a c i d s y n t h e t a s e . S c i e nc e . 1 7 3 : 2 4 3( 1 9 7 3 ) .

1 2 . J a c k s o n , W. T . R e g u l a t i o n o f m i t o s i s : I I I . C y t o -l o g i c a l e f f e c t s o f 2 , 4 , 5 - T a n d o f d io x i n c o n t a m -

i n a n t s i n 2 , 4 , 5 - T f o r m u l a t i o n s , J . C e l l S c i . 1 0 :1 5 ( 1 9 7 2 ) .

1 3 . A l l e n , J . R . , a n d C a r s t e n s , L . A . L i g h t a n de l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s i n Macacam u l a t t a m o n k e y s f e d t o x i c f a t . Am. J . V e t .R e s . 2 8 : 1 5 1 3 ( 1 9 6 7 ) .1 4 . H e r b i c i d e A s s e s s m e n t C o m m i s s io n. B a c k g r o u n dM a t e r i a l R e l e v a n t t o P r e s e n t a t i o n s a t t h e 1 9 7 0

A n n u a l M e e t i n g o f t h e AAAS a n d P r e l i m i n a r yR e p o r t o f t h e H e r b i c i d e A s s e s s m e nt C o m m i s -s i o n ; b o t h r e p r i n t e d i n C on g r e s s i o n a l R e c o r d1 1 8 ( 3 2 ) : S 3 2 2 6 - 3 2 3 3 M a r c h 3 , 1 9 7 2 .1 5 . B u u - H o i , N . P . , e t a l . O r g a n s a s t a r g e t s o fd i o x i n i n t o x i c a t i o n , N a t u r w i s s . 5 9 : 1 7 4 ( 1 9 7 2 ) .1 6 . D a v r i n g , L . , a n d S u n n e r , M . C y t o g e n e t i c e f -f e c t s o f 2 , 4 , 5 - t r i c h l o r o p h e n o x y a c e t i c a c i d o no o g e n e s i s a n d e a r l y e m b r y o g e n e s i s i n D r o s o p h i l am e l a n o g a s t e r . H e r e d i t a s 6 8 : 1 1 5 ( 1 9 7 1 ) .

Navy Chemicals in Potable Water 2

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