U.S. DEPARTMENT OF COMMERCE
                     National Technical Information Service
                     PB-262 897
Acute Toxicity of Selected
Organic Compounds  to
Fathead Minnows
Environmental Research Lab, Duluth, Minn

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EPA-600/3-76-097
October  1976
EicoSogbcl
        Scries
       GRGANii  CO
                                                         -* "> £
                                                         ur
                                                                   SF" -:>",;rch mrf Oeveiopment
                                                  i.5  j'-'^iitiTo^tal Protection Agency
                                                          Ouiuth, Minnesota 55804

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                  RESEARCH REPORTING SERIES

 Research reports of the Office of Research and Development, U.S. Environmental
 Protection Agency,  have been grouped into five series. These five broad
 categories were established to facilitate further development and application of
 environmental technology. Elimination of traditional grouping was consciously
 planned to foster technology transfer and a maximum interface in related fields.
 The five series are:

      1.    Environmental Health Effects Research
      2.    Environmental Protection Technology
      3.    Ecological Research
      4.    Environmental Monitoring
      5.    Socioeconomic Environmental Studies

 This report has been assigned to the ECOLOGICAL RESEARCH series. This series
 describes  research on the effects  of  pollution on humans, plant and animal
 species, and materials. Problems are assessed for their long- and short-term
 influences. Investigations include formation, transport, and  pathway studies to
 determine the fate of pollutants and their effects. This work provides the technical
 basis for setting standards to minimize undesirable changes in living organisms
 in the aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.

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                                   TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
1. REPORT NO.
  EPA-600/3-T6-09T
                             2.
4. TITLE AND SUBTITLE
  ACUTE TOXICITY  OF SELECTED ORGANIC  COMPOUNDS
  TO FATHEAD MINNOWS
                                                           3. RECIPIENT'S ACCESSION-NO.
                                                           5. REPORT DATE
                                                            October 1976
 (Issuing Date)
                                                           6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
  Vincent R. Mattson, John W. Arthur,  and
  Charles T. Walbridge
                                                           8. PERFORMING ORGANIZATION REPORT NO.
                                                           10. PROGRAM ELEMENT NO.
                                                              1BA608
9. PERFORMING ORGANIZATION NAME AND ADDRESS
  Environmental  Research Laboratory
  U.S. Environmental Protection Agency
  6201 Congdon Boulevard
  Duluth, Minnesota  5580^
                                                           11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
  Environmental  Research Laboratory
  Office of Research and Development
  U.S. Environmental Protection Agency
  Duluth, Minnesota  5580U	
                                                           13. TYPE OF REPORT AND PERIOD COVERED
                                                             Final (Feb.  1973-July 197M
                                                           14. SPONSORING AGENCY CODE
                                                             EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
   Static nonrenewal laboratory bioassays vere conducted vith 26 organic  compounds
   commonly used by industry.   The selected compounds represented the five  following
   chemical classes:  acids,  alcohols, hydrocarbons,  ketones and aldehydes,  and phenols.
   Juvenile fathead minnows  (Pimephales promelas) were the test animal, and test
   duration was limited to 96 hr.   Lake Superior water served as the diluent source
   for all test compounds.  Additional tests were conducted with a reconstituted
   diluent water for five compounds.   The alcohol compounds were generally  10 to 100
   times less toxic than chemicals tested in the other four chemical classes.
   Compounds in the other four classes had a similar  toxicity range.  The most lethal
   chemical tested was pentachlorophenol (96-hr LC50  of 0.6 mg/1.) and the  least
   lethal was ethanol (96-hr  LC50  of 13,^80 mg/1.).   Most of the minnow deaths occurred
   during the first 2k hr.  Comparative chemical tests in the two diluent waters gave
   similar lethal responses.
17.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
                                              b.lDENTIFIERS/OPEN ENDED TERMS  C. COSATI Field/Group
  Organic  compounds
  Bioassay
  Minnows
                                               Acute toxicity
                                               Reconstituted water
                                               Juvenile  fathead minnows
  6A
  6C
  7C
 3. DISTRIBUTION STATEMENT

  RELEASE TO  PUBLIC
                                              19. SECURITY CLASS (ThisReport)
                                               UNCLASSIFIED
21, NO. OF PAGES
                                              20. SECURITY CLASS (Thispage)
                                               UNCLASSIFIED
EPA Form 2220-1 (9-73)
                                                                    •fr U.S.GOWB1tt
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                                   DISCLAIMER

This report has been reviewed by the Environmental Research Laboratory-Duluth,
U.S. Environmental Protection Agency, and approved for publication.  Mention
of trade names or commercial products does not constitute endorsement or
recommendation for use.
                                       11

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                                       EPA-600/3-76-097
                                       October 1976
ACUTE TOXICITY OF SELECTED ORGANIC COMPOUNDS

             TO FATHEAD MINNOWS
             Vincent Ru Mattson
               John W. Arthur
            Charles T0 Walbridge
      Environmental Research Laboratory
          Duluth,  Minnesota
       ENVIRONMENTAL RESEARCH LABORATORY
     OFFICE OF RESEARCH AND DEVELOPMENT
    U.S. ENVIRONMENTAL PROTECTION AGENCY
          DULUTH, MINNESOTA  5580U

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                                 FOREWORD

Our nation's freshwaters are vital for all animals and plants, yet our
diverse uses of water	for recreation, food, energy, transportation, and
industry	physically and chemically alter lakes, rivers, and streams.  Such
alterations threaten terrestrial organisms, as veil as those living in water.
The Environmental Research Laboratory in Duluth, Minnesota develops methods,
conducts laboratory and field studies, and extrapolates research findings

    —to determine how physical and chemical pollution affects
      aquatic life

    —to assess the effects of ecosystems on pollutants

    —to predict effects of pollutants on large lakes through
      use of models

    —to measure bioaccumulation of pollutants in aquatic
      organisms that are consumed by other animals, including
      man

This report describes the short-term toxicity of 26 organic  compounds to
fathead minnows.  The laboratory tests were limited to U-days1 duration.
                                     Donald  I. Mount, Ph.D.
                                     Director
                                     Environmental Research  Laboratory
                                     Duluth, Minnesota
                                     111

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                                   ABSTRACT

Static nonrenewal laboratory bioassays were conducted with 26 organic
compounds commonly used by industry.  The selected compounds represented
the five following chemical classes:  acids, alcohols, hydrocarbons,
ketones and aldehydes, and phenols.  Juvenile fathead minnows (Pimephales
promelas) were the test animal, and test duration was limited, to 96 hr.
Lake Superior water served as the diluent source for all test compounds.
Additional tests were conducted with a reconstituted diluent water for five
compounds.  The alcohol compounds were generally 10 to 100 times less toxic
than chemicals tested in the other four chemical classes.  Compounds in the
other four classes had a similar toxicity range.  The most lethal chemical
tested was pentachlorophenol (96-hr LC50 of 0.6 mg/1.) and the least lethal
was ethanol (96-hr LC50 of 13,^80 mg/1.).  Most of the minnow deaths
occurred during the first 2k hr.  Comparative chemical tests in the two
diluent waters gave similar lethal responses.

This report was submitted in partial fulfillment of Project Number 16-AAE
by the Environmental Research Laboratory-Duluth.  Experimental work was
completed in July
                                    iv

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                             CONTENTS




                                                                    Page




Foreword	iii




Abstract	*	 . iv




Acknowledgments  	 vi




I      Introduction	»	  1




II     Conclusions	2




III    Recommendations	3




IV     Methods and Materials.	h




V      Results and Discussion 	  7




VI     References	11

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                                ACKNOWLEDGMENTS
The authors thank Dr. Oilman D. Veith for assistance during this study.
                                   VI

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                                  SECTION I
                                 INTRODUCTION

Industrial production estimates for 1973 of the top 50 inorganic and organic
chemicals (based on volume) exceeded 1*00 billion pounds.  Production
volumes of the organics, as a group, increased over previous years
(Anderson, 197*0 •  There has been and still exists a demand for information
concerning the harmful effects of organic chemicals on aquatic life.  The
Battelle Columbus Laboratories (1971) and Kemp et_al. (1973) have compiled
extensive lists of the effects of chemicals on aquatic life.  MacPhee and
Ruelle (1969) studied the effects of 1,888 chemicals on four fish species,
but the tests were limited to 2k hours' duration and to chemical concentrations
of 10 mg/1. or less.  Canadian investigators have published comprehensive
reviews on the toxicity of individual chemicals commonly found in various
effluents.  Clarke (197*0 summarized the toxicity of chemical components from
inorganic chemical industries.  The toxicity of effluents containing large
inclusions of organic chemicals has been reviewed by Hunka (197*0 for the
plastics industries, Thompson (197*0 for the textiles industries, and Smith
(197*0 for petrochemical plants.

The Environmental Research Laboratory-Duluth received from Donaldson (1972) a
list of 257 organic compounds that he identified from industrial wastewaters,,
Twenty-six compounds were selected for testing based on their reported importance
to industry (Kirk and Othmer, 1963-1969; Hawley, 1971) and on their use as parent
compounds from which derivatives are commonly made.  Since very little
information was available on the comparative toxicity of chemical groups,
the 26 compounds were grouped into the following five classes:  alcohols,
acids, hydrocarbons, ketones and aldehydes, and phenols.  This report
then gives the concentrations of each chemical lethal to fathead minnows
during short-term 96-hr static tests.

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                                   SECTION II
                                  CONCLUSIONS

1.   The alcohols tested were generally 10 to 100 times less toxic to fathead
     minnows than the acids, hydrocarbons, ketones and aldehydes, and phenols.
     A loss in equilibrium generally preceded death in all chemical groups
     groups tested except the acids.

2.   Pentachlorophenol was the most toxic compound tested and ethanol the
     least toxic.

3.   Most of the deaths during the It-day tests occurred within the first
     2k hr and may have been related to the methods employed.

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                                  SECTION III
                                RECOMMENDATIONS

Information is lacking on the individual long-term consequences to
aquatic life of the chemicals tested in this study.  Industrial effluents
are typically represented by a mixture of many chemicals.   Studies are
therefore needed on their combined interactive effects  on  both a short-
and long-term exposure basis.

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                                   SECTION  IV
                             METHODS AND MATERIALS

Juvenile  fathead minnows obtained  from the Environmental Research Laboratory-
Duluth  cultures served as the test animal.  All  fish used for the test vere
from k  to  8 veeks old and varied in length from  1.1 to  3.1 cm.  Fish were
acclimated in flowing water 11 cm  deep in  a holding trough (3&0 cm long by
58 cm wide by 31 cm deep) for at least 1*8  hr before the tests.  Water
temperature was 18-22° C.  During  acclimation the fish were fed Glencoe
trout chow no. 3 granules.

The organic compounds tested and the supplier are given in Table 1.  All
compounds  were reagent-grade chemicals.  Test solutions were prepared by
adding  a weighed amount of chemical to a 9-1. glass carboy containing
k I. of Lake Superior water.  Solution components were thoroughly mixed
by shaking and Were then poured into two glass battery Jars for preparation of
the toxicant test concentrations.  All toxicant  concentrations are
nominal; none were analyzed to determine concentration levels.  A
fiberglass trough of the same dimensions and water depth as used for
acclimation served as a water bath for maintaining the test solutions at
l8°-22° C.

The static tests were conducted in 3-1. cylindrical glass battery jars
containing 2 1. of test water.  Exploratory tests were conducted
for many of the compounds to determine the lethal range before attempting
a definitive test.  Three to five  widely spaced  test concentrations
differing by a factor of 10 or more and a control were used.  These tests
were usually limited to 2k hours'  duration.

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               TABLE 1.  ORGANIC COMPOUNDS TESTED AND SOURCE OF  SUPPLY
     Chemical
                              Formula
                                                               Company
Acetic acid




Acetophenone




Adlplc acid




Benzyl alcohol




1-Butanol




d-Camphor




Caprolc acid




p-Cresol




Cyclohexane




Cyclohexanol




Ethanol




Eugenol




Furfural




Indan




Isopropanol




1-Methylnapthalene




m-Nitrotoluene




Olelc acid




Pentachlorophenol




Phenol




Quinoline




Styrene monomer




Valeric acid




Vanillin




Xylene




3,4 Xylenol
CH3COOH




C6H5COCH3




COOH(CH2).,COOH




C6H5CH2OH




CH3(CH2)2CH2OH





CloH16°



CH3(CH2),,COOH




CH o Cc HI, OH




C6H12




C6HUOH




C2H5OH




C3H5C6H3(OH)OCH3




C,,H3OCHO




CHCHCHCHCpCH2 CH2 C*H2




(CH3)2CHOH
CH3(CH2)7CH:CH(CH2)7COOH




C6C15OH




C6H5OH




CHCHCHCH^CNCHCHdH




C6H5CH:CH2




CH3(CH2)3COOH




(CH30)(OH)C6H3CHO




C6HM(CH3)2




(CH3)2C6H3OH
Burklck and Jackson Lab. Inc.




Curtin Matheson Scientific Inc.




Curtin Matheson Scientific Inc.




Curtin Matheson Scientific Inc.




Burklck and Jackson Lab. Inc.




Curtin Matheson Scientific Inc.




Aldrich Chemical Co.




Curtin Matheson Scientific Inc.




Burkick and Jackson Lab. Inc.




Curtin Matheson Scientific Inc.




U.S. Industrial Chemical Co.




Aldrich Chemical Co.




Curtin Matheson Scientific Inc.




Curtin Matheson Scientific Inc.




Burkick and Jackson Lab. Inc.




Curtin Matheson Scientific Inc.




Curtin Matheson Scientific Inc.




Coleman and Bell




Aldrich Chemical Co.




Mallinckrodt Chemical Works




Curtin Matheson Scientific Inc.




Curtin Matheson Scientific Inc.




Aldrich Chemical Co.




Curtin Matheson Scientific Inc.




Aldrich Chemical Co.




Aldrich Chemical Co.
a
 From Hawley  (1971).

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Definitive tests were started by placing 10 fish into each battery jar
so that 20 individuals were tested at each concentration.  A glass cover
was placed over each jar to reduce evaporation.  The test waters were not
aerated.  Fish were not fed during the tests.

Complete immobilization of the fish was considered as the biological
endpoint and equated with death.  Fish mortality was measured after 1,
2H, U8, and 96 hr.  Standard graphical procedures were followed for
determining concentrations that would result in 50% mortality (American
Public Health Association, 1971 )<»

Five organic compounds were also tested in soft reconstituted water.  The
objective was to compare the toxic response in two different diluent
waters.  The animals were acclimated in Lake Superior water.
The reconstituted water was prepared by adding NaHCO , CaSO • 2H_0, MgSO. ,
and KC1 salts to distilled water in quantities specified by the Committee
on Methods for Toxicity Tests with Aquatic Organisms (1975).  Replicate
tests were conducted with vanillin in both diluent waters.  Comparative
tests were also conducted with vanillin in reconstituted water for
minnows acclimated in lake and reconstituted waters.  Vanillin was chosen
for these additional tests because of its low handling hazard to human beings
(Hawley, 1971).

Analyses of the test waters for dissolved oxygen and pH were made at the
beginning and one or two times in the course of the test,.  Dissolved
oxygen measurements were made with a YSI oxygen meter, pH measurements
with a Corning pH meter.  Water temperature was measured daily in two of
the test containers with a mercury thermometer.

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                                   SECTION V
                            RESULTS AND DISCUSSION

Concentrations toxic to the minnows in Lake Superior water and in reconstituted
water are presented in Tables 2 and 3, respectively.  For five chemicals the
LC50 values did not change by more than 10/? after 1 hr of exposure (l-butanolf
isopropanol, cyclohexane, xylene, and eugenol).  The 2k- and 96-hr LC50 levels
for IT of 26 compounds were alike.  Many of the chemicals noticeably affected
minnow equilibrium before death.  Loss of equilibrium frequently disappeared
for minnows surviving beyond 2k~kQ hr.  Equilibrium losses were not generally
observed for the acid compounds tested.  The lethal and locomotor observations
can be related to the static method employed and the compound's vapor pressure.
These results support the recommendation by the American Public Health
Association (19T1> P. 570) that test solutions of volatile or unstable
compounds be renewed every 2k hr or less.

Toxicity results obtained in reconstituted water for one organic compound
from four of the five classes were nearly identical to those obtained in
Lake Superior diluent water (Tables 2 and 3).  Toxic responses by fathead
minnows when acclimated in both lake and reconstituted water before tests
with vanillin were also similar.  Except for the 1-hr LC50 values, all
remaining replicate vanillin LC50 values in, both diluent waters were similar.

Tests with dissolved oxygen concentrations less than k.Q mg/1. and pH values
less than 5.9, are denoted in Tables 2 and 3.  Earlier work has
shown that at these concentrations low oxygen and pH themselves can cause
adverse effects on fathead minnows in long-term toxicity tests (Brungs,
1971; Mount, 1973).

Alcohols, with the exception of benzyl alcohol, were 10 to 100 times
less toxic than compounds tested in the other four chemical
classes (Tables 2 and 3).  Toxicity of the alcohols increased with increasing
                                       7

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  TABLE  2.   LETHAL  CONCENTRATIONS  OF 26  ORGANIC COMPOUNDS TO FATHEAD


                     MINNOWS  IN  LAKE  SUPERIOR WATER
Test
chemical
Acids
Aceticb
Adipicb
Caproic
Oleic3
Valeric
Alcohols
Benzyl
1-Butanol
Cyclohexanol
Ethanol3
Isopropanol
Hydrocarbons
Cyclohexane
Indan
1-Methylnapthalene
m-Nitrotoluene
Quinoline
Styrene
Styrenec
Xylene
Ketones and Aldehydes
Ace tophenone
d-Camphor
Furfural
Vanillin-Test la
Vanillin-Test 2a
Phenols
p-Cresola
Eugenol
Pentachlorophenol
Phenolb
3,4 Xylenol
LC50 concentration (in mg/1.)
1-hr

>315
>300
140
> 1,000
>100

770
1,950
1,550
> 18, 000
11,830

95
39
39
43
78
100
40
46

>200
145
> 50
>173
370

>30
24
8
>50
>20
24-hr

122
172
88
285
>100.

770
1,950
1,033
>18,000
11,160

93
14
9
30
46
32
30
42

>200
112
48
131
125

26
24
0.6
>50
>20
48-hr

92
114
88
252
77

770
1,950
1,033
13,480
11,130

93
14
9
30
46
32
29
42

163
111
37
123
116

21
24
0.6
>50
15
72-hr

88
97
88
205
77

480
1,950
1,033
13,480
11,130

93
14
9
30
46
32
29
42

158
110
32
121
112

21
24
0.6
33
14
96-hr

88
97
88
205
77

460
1,910
1,033
13,480
11,130

93
14
9
30
46
32
29
42

155
110
32
121
112

19
24
0.6
32
14
 Dissolved oxygen measured £4.0 mg/1. during test.


 pH measured £.5.9 units during test.

£
 Acetone added to diluent water.

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     TABLE 3.   LETHAL CONCENTRATIONS OF FIVE ORGANIC COMPOUNDS TO


                FATHEAD MINNOWS IN RECONSTITUTED WATER
Test
chemical
Acetic acid
1-Butanol
Cyclohexane
Furfural
Vanillin-Test ia'C
Vanillin-Test 2a'C
Vanillina'd
LC50 concentration (in mg/1.)
1-hr
175
1,9^0
126
>50
3U8
>1T3
>173
2U-hr
106
1,9^0
117
>50
100
127
125
148-hr
106
1,9^0
117
37
97
121
116
72-hr
79
1,9^0
117
33
88
121
116
96 -hr
79
1,9^0
117
32
88
121
116
^Dissolved oxygen measured 
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molecular weight.  Oleic acid additions to the diluent lake water did not
completely dissolve.  Hawley (1971) lists this compound as insoluble in
water.  Actual LC50 values for oleic acid may be lower.  The toxicity
values: obtained for styrene were  similar with and without the additions
of acetone (at a concentration of  500 mg/1.) in the diluent lake water
except after 1-hr exposure (Table  l).  Pentachlorophenol was the most
toxic compound tested and had a lethal concentration  50 times greater
than phenol and about 25 times greater than 3,^ dimethyl phenol (xylenol).
Kopperman et_ al_. (197*0» in comparable tests with Daphnia magna, found
that U-chlorophenol was 37 times more toxic than phenol and
concluded that phenol toxicity was increased significantly with increasing
halogen content.

The following chemical concentrations were within 50% of the
lethal concentrations reported (as TLm or LC50 values) in the
toxicological reviews of Battelle  Columbus Laboratories (1971), Hunka
(197*0 > and Smith (197*0 for 96-hr fish static bioassays:  acetic,
adipic, and caproic acids, ethyl alcohol, cyclohexane, styrene, xylene,
eresol, furfural, and phenol.  Lethal levels for cyclohexane were two to
three times higher than cited in Hunka (197*0 in "clear" water.  Time
required to produce death in a study by MacPhee and Ruelle (1969) for 10
mg/1. concentrations of furfural and xylenol were three to four times
less than in our study.  Test water temperature was reported to be about
12° C.  No further comparable information pertaining to fish was found for the
remaining chemicals tested.

The U.S. Environmental Protection Agency (197*0 assembled a list of
chemicals regarded as hazardous substances based on their reported short-
term toxicity and potential for being discharged or spilled into
waterways.  Ten of the compounds tested in our study were on this list.
It may never be possible to evaluate with short-term tests the toxicity
of  all industry-related organic compounds and derivatives to aquatic life.
Future work could be pointed towards methodology which may predict toxicity.
                                      10

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                                  SECTION VI

                                  REFERENCES
American Public Health Association.  1971-  Standard methods for the
examination of water and wastewater.  13th ed.  Washington, D.C.  87^ p.

Anderson, E. V.  197^.  Growth slows in top 50 chemicals' output.  Chem.
Eng. News 52:10-13.

Battelle Columbus Laboratories.  1971.  Water quality criteria -
Volume 3.  Effects of chemicals on aquatic life,,  U.S. Environmental
Protection Agency, Water Pollution Control Research Series, May,
18050GWV05/71.  PP. Al-166 and Bl-236.

Brungs, W. A.  1971.  Chronic effects of low dissolved oxygen
concentrations on the fathead minnow (Pimephales promelas).  J. Fish.
Res., Board Can. 38:1119-1123°

Clarke, R. M.  197^.  A summary of toxicity information for major effluent
components from inorganic chemical industries.  Environment Canada, Dept.
Environ. Fish,, and Marine Service, Tech. Rep. Series No0 CEN/T-7^-9.  27 p.

Donaldson, W. T0  1972.  Memorandum to D. I. Mount, National Water Quality
Laboratory  (now the Environmental Research Laboratory) Duluth, Minn.  December 8.

Hawley, G. G.  1971-  The condensed chemical dictionary.  8th ed.  Van
Nostrand Reinhold Company, New York, N.Y. 971 p.

Hunka, D. L0  197^.  The effects of effluents from the Canadian plastics
industry on aquatic organisms.  A literature review.  Environment
Canada, Depto Environ. Fish, and Marine Service, Tech. Rep0 No. U73.
6h p.

Kemp, H. T., R. L. Little, V. L. Holoman, and R. L. Darby.  1973.  Water
quality criteria data book - Volume 5.  Effects of chemicals on aquatic
life.  U.So Environmental Protection Agency, Water Pollution Control
Series, September, 18050HLA 09/73.  pp. A1-W9,,

Kirk, R. E. and D. F. Othmer»  1963-1969.  Encylopedia of chemical
technology.  22 volumes.  John Wiley & Sons, Inc., New York, N.Y.

Kopperman, A. L., R. M. Carlson, and R. Caple«  197^«  Aqueous chlorination
and ozonation studies.  I.  Structure-toxicity correlations of phenolic
compounds to Daphnia magna.  Chem. Biol. Interactions 9:2U5-251o
                                        11

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MacPhee, C., and C. Ruelle.  1969.  Lethal effects of 1,888 chemicals upon
four species of fish from western North America.  Univ. Idaho, Forest,
Wildlife, and Range Exp. Sta., Moscow, Idaho, Bull. 3.  112 p.

Mount, D. I.  1973.  Chronic effect of low pH on fathead minnow survival,
growth,, and reproduction,,  Water Res. 7:987-993.

Smith, A. L.  197**.  The effects of effluents from the Canadian petrochemical
industry on aquatic organisms.  A literature review.  Environment Canada,
Depto Environ. Fish, and Marine Service, Tech. Rep. No. 1+72.  68 p.

The Committee on Methods for Toxicity Tests with Aquatic Organisms.  1975.
Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians.
U.S. Environmental Protection Agency, Duluth, Minn»  Ecological Research Series
EPA-660/3-75-009.  67 p.

Thompson, B.  197^.  The effects of effluent from the Canadian textile
industry on aquatic organisms.  A literature review.  Environment Canada,
Dept. Environ. Fish, and Marine Service, Tech. Rep0 No. U89.  99 p.

U.S. Environmental Protection Agency.  197^.  Designation and determination
of removability of hazardous substances from water.  Proposed rules.  Federal
Register 39:30U66-30Vri.
                                      12

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                      COM-75-11070/PAT 48 p.  PC$4.00/MF$3.00

                      Data Base Directions. The Next Steps
                      PB-258 103/PAT 177 p.  PC$7.50/MF$3.00

                      Comparative Study of Various Text Editors and Formatting
                      Systems
                      ADA-029 050/PAT  93 p.  PC Not Available/MF$3.00

                      Explaining Energy: A Manual of Non-Style for the Energy
                      Outsider Who Wants In
                      LBL-4458/PAT  78 p.  PC$4.50/MF$3.00

                      A Survey of State Legislation Relating to Solar Energy
                      PB-258 235/PAT 166 p.  PC$6.75/MF$3.00

                      Cost Estimating Handbook for Transfer, Shredding and
                      Sanitary Landfilling of Solid Waste
                      PB-256 444/PAT 85 p.  PC$5.00/MF$3.00

                      Coal Liquefaction Design Practices Manual
                      PB-257541/PAT 372 p.  PC$10.50/MF$3.00
HOW TO ORDER
  When you indicate the  method  of  pay-
ment, please note if a purchase order is not
accompanied by payment, you will be billed
an additional $5.00 ship and bill charge. And
please include the card expiration date when
using American Express.

  Normal delivery time  takes three to five
weeks. It is vital that you order by number
or your order will be manually filled, insur-
ing a delay. You can opt for airmail delivery
for $2.00 North  American continent; $3.00
outside North American continent charge per
item. Just check the Airmail  Service box. If
you're really pressed for time, call the NTIS
Rush Handling Service (703)557-4700. For a
$10.00 charge per item, your order will be
airmailed within 48 hours. Or, you can  pick
up your order in the Washington  Informa-
tion Center & Bookstore or at our Springfield
Operations Center within 24 hours for a
$6.00 per item charge.
  You may also place your order by tele-
phone or if you have an NTIS Deposit Ac-
count or an American Express card order
through TELEX. The order desk number is
(703) 557-4650 and the TELEX number is
89-9405.
  Thank you for your interest in NTIS. We
appreciate your order.
METHOD OF PAYMENT
D  Charge my NTIS deposit account no.
D  Purchase order no.	
    Check enclosed for $_
                                                          NAME	
    Bill me. Add $5.00 per order and sign below. (Not avail-
    able outside North American continent.)                  ADDRESS.
    Charge to my American Express Card account number
                                                          CITY. STATE. ZIP.
 Card expiration dale-

 Signature 	
    Airmail Services requested


    Clip and mail to:
    National Technical Information Service
    U.S.  DEPARTMENT OF COMMERCE
    Springfield. Va. 22161
    (703) 557-4650 TELEX 89-9405
Item Number




Quantity
Paper Copy
(PC)




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(MF)




Unit Price*




All prices subject to change. The prices Sub Total
above are accurate as of 4/77 Additional Charge
Enter Grand Total
Total Price"








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