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    INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY



    ENVIRONMENTAL HEALTH CRITERIA 84






    2,4-DICHLOROPHENOXYACETIC ACID (2,4-D) - ENVIRONMENTAL ASPECTS






    







    This report contains the collective views of an international group of
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    policy of the United Nations Environment Programme, the International
    Labour Organisation, or the World Health Organization.

    Published under the joint sponsorship of
    the United Nations Environment Programme,
    the International Labour Organisation,
    and the World Health Organization

    World Health Orgnization
    Geneva, 1989


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CONTENTS

ENVIRONMENTAL HEALTH CRITERIA FOR 2,4-DICHLOROPHENOXYACETIC ACID 
(2,4-D) - ENVIRONMENTAL ASPECTS 

 1. SUMMARY AND CONCLUSIONS 

     1.1. Uptake, accumulation, elimination, and biodegradation       
     1.2. Toxicity to microorganisms  
     1.3. Toxicity to aquatic organisms   
     1.4. Toxicity to terrestrial organisms   
     1.5. Effects of 2,4-D in the field   

 2. PHYSICAL AND CHEMICAL PROPERTIES    

     2.1. Synthesis of 2,4-D  
     2.2. Important chemical reactions of 2,4-D   
     2.3. Volatility of 2,4-D derivatives 

 3. SOURCES OF ENVIRONMENTAL POLLUTION  

     3.1. Production of 2,4-D herbicides  
     3.2. Uses                
     3.3. Disposal of wastes  

 4. UPTAKE, ACCUMULATION, ELIMINATION, AND  BIODEGRADATION      

     4.1. Biodegradation      
     4.2. Uptake and accumulation by organisms    
          4.2.1. Laboratory studies  
          4.2.2. Field studies   
     4.3. Elimination     

 5. TOXICITY TO MICROORGANISMS  

     5.1. Aquatic microorganisms  
     5.2. Soil microorganisms 

 6. TOXICITY TO AQUATIC ORGANISMS   

     6.1. Toxicity to aquatic invertebrates   
          6.1.1. Short-term toxicity 
          6.1.2. Behavioural effects 
     6.2. Toxicity to fish    
          6.2.1. Effect of formulation on short-term toxicity to fish    
                  6.2.1.1 Tolerance and potentiation  
          6.2.2. No-observed-effect-levels in short-term tests with fish
          6.2.3. Species differences in short-term toxicity to fish  
          6.2.4. Toxicity to early life-stages of fish   
          6.2.5. Long-term toxicity to fish  
          6.2.6. Behavioural effects on fish 
          6.2.7. Effects of environmental variables on toxicity to fish 
          6.2.8. Special studies on fish 
     6.3. Toxicity to amphibians  

 7. TOXICITY TO TERRESTRIAL ORGANISMS   

     7.1. Toxicity to terrestrial invertebrates   
     7.2. Toxicity to birds
          7.2.1. Toxicity to birds' eggs
          7.2.2. Toxicity to birds after short-term and long-term dosing
          7.2.3. Special studies on birds    
     7.3. Toxicity to non-laboratory mammals  

 8. ECOLOGICAL EFFECTS FROM FIELD APPLICATION   

 9. EVALUATION          

     9.1. Aquatic organisms   
     9.2. Terrestrial organisms   

10. RECOMMENDATIONS FOR FURTHER RESEARCH    

REFERENCES  


WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR
2,4-DICHLOROPHENOXYACETIC ACID (2,4-D) - ENVIRONMENTAL ASPECTS

 Members

Dr L.A.  Albert, Director, Environmental Pollution Programme, National
   Institute for Research on Biotic Resources, Veracruz, Mexico
Mr H.   Craven,  Ecological  Effects  Branch,   Office  of  Pesticides
   Programs, US Environmental Protection Agency, Washington DC, USA
Dr A.H.  El-Sebae,  Division  of  Pesticide  Toxicology,  Faculty   of
   Agriculture, Alexandria University, Alexandria, Egypt
Dr J.W.  Everts,  Department  of Toxicology,  Agricultural University,
   Wageningen, Netherlands
Dr W.  Fabig,  Fraunhofer  Institute for  Environmental  Chemistry and
   Ecotoxicology,   Schmallenberg-Grafschaft,   Federal  Republic   of
   Germany
Dr R. Koch, Division of Toxicology, Research Institute for Hygiene and
   Microbiology, Bad Elster, German Democratic Republic  (Chairman)
Dr Y.  Kurokawa,  Division  of Toxicology,  Biological Safety Research
   Centre, National Institute of Hygienic Sciences, Tokyo, Japan
Dr E.D.  Magallona,  Pesticide  Toxicology and  Chemistry  Laboratory,
   University of the Philippines at Los Banos, College of Agriculture,
   Laguna, Philippines
Professor  P.N.  Viswanathan,  Ecotoxicology Section,  Industrial Toxi-
   cology Research Centre, Lucknow, India

 Observers

Dr M.A.S.  Burton,  The  Monitoring and  Assessment  Research  Centre,
   London, United Kingdom
Dr I.  Newton,  The  Institute  of  Terrestrial  Ecology,  Monks  Wood
   Experimental Station, Huntingdon, United Kingdom

 Secretariat

Dr S.  Dobson,  The  Institute  of  Terrestrial  Ecology,  Monks  Wood
   Experimental Station, Huntingdon, United Kingdom  (Rapporteur)
Dr M.  Gilbert,  International  Programme on  Chemical  Safety,  World
   Health Organization, Geneva, Switzerland  (Secretary)
Mr P.D.  Howe,  The  Institute  of  Terrestrial  Ecology,  Monks  Wood
   Experimental Station, Huntingdon, United Kingdom

NOTE TO READERS OF THE CRITERIA DOCUMENTS


    Every effort has been made to present information in  the  criteria
documents  as  accurately as  possible  without unduly  delaying  their
publication.   In the interest of all users of the environmental health
criteria  documents, readers are  kindly requested to  communicate  any
errors  that may  have occurred  to the  Manager of  the  International
Programme  on  Chemical  Safety,  World  Health  Organization,  Geneva,
Switzerland,  in order that they  may be included in  corrigenda, which
will appear in subsequent volumes.



                    *      *      *



    A detailed data profile and a legal file can be obtained  from  the
International  Register  of  Potentially Toxic  Chemicals,  Palais  des
Nations, 1211 Geneva 10, Switzerland (Telephone no.  988400 - 985850).

ENVIRONMENTAL HEALTH CRITERIA FOR 2,4-DICHLOROPHENOXYACETIC ACID 
(2,4-D) - ENVIRONMENTAL ASPECTS

    A   WHO   Task  Group   on   Environmental  Health   Criteria   for
2,4-Dichlorophenoxyacetic  acid (2,4-D) - Environmental Aspects  met at
the  Institute of Terrestrial Ecology, Monks Wood, United Kingdom, from
14  to  18 December  1987.  Dr I.  Newton welcomed the  participants on
behalf of the host institution, and Dr M. Gilbert opened the meeting on
behalf   of  the  three   co-sponsoring  organizations  of   the   IPCS
(ILO/UNEP/WHO).  The Task Group reviewed and revised the draft criteria
document and made an evaluation of the risks for the  environment  from
exposure to 2,4-D.

    The first draft of this document was prepared by Dr S.  Dobson  and
Mr  P.D.  Howe, Institute  of Terrestrial Ecology.   Dr M. Gilbert  and
Dr  P.G. Jenkins, both members  of the IPCS Central  Unit, were respon-
sible for the overall scientific content and editing, respectively.



                    *      *       *



    Partial  financial  support for  the  publication of  this criteria
document  was kindly provided by the United States Department of Health
and Human Services, through a contract from the National  Institute  of
Environmental  Health Sciences, Research Triangle Park, North Carolina,
USA - a WHO Collaborating Centre for Environmental Health Effects.

INTRODUCTION

    There  is  a  fundamental  difference  in  approach   between   the
toxicologist  and the ecotoxicologist  concerning the appraisal  of the
potential  threat posed by  chemicals.  The toxicologist,  because  his
concern  is with  human health  and welfare,  is preoccupied  with  any
adverse  effects on  individuals, whether  or not  they  have  ultimate
effects  on performance or survival.  The ecotoxicologist, in contrast,
is  concerned primarily with  the maintenance of  population levels  of
organisms in the environment.  In toxicity tests, he is  interested  in
effects on the performance of individuals - in their  reproduction  and
survival - only insofar as these might ultimately affect the population
size.  To him, minor biochemical and physiological effects of toxicants
are irrelevant if they do not, in turn, affect reproduction, growth, or
survival.

    It  is the aim of this document to take the ecotoxicologist's point
of  view  and  consider effects  on  populations  of organisms  in  the
environment.   The  risk  to human  health  of  the use  of  2,4-D  was
evaluated  in  Environmental  Health Criteria  29: 2,4-Dichlorophenoxy-
acetic  acid (WHO, 1984).   This document did  not consider effects  on
organisms  in the environment, but did consider environmental levels of
2,4-D  likely to arise from recommended uses.  No attempt has been made
here  to reassess the human  health risk; the interested  reader should
refer  to the original document, which contains the relevant literature
in this area.

    This  document,  although  based  on  a  thorough  survey  of   the
literature,  is not intended to be exhaustive in the material included.
In  order to  keep the  document concise,  only those  data which  were
considered to be essential in the evaluation of the risk posed by 2,4-D
to the environment have been included.

    The term bioaccumulation indicates that organisms take up chemicals
to  a  greater concentration  than that found  in their environment  or
their  food.   `Bioconcentration  factor'  is  a  quantitative  way  of
expressing  bioaccumulation:   the ratio  of  the concentration  of the
chemical  in the organism to  the concentration of the  chemical in the
environment or food.  Biomagnification refers, in this document, to the
progressive accumulation of chemicals along a food chain.

1.  SUMMARY AND CONCLUSIONS

    2,4-D  is a selective herbicide which kills broad-leaved plants but
not grasses or conifers.  Its chemical structure is a modification of a
naturally  occuring plant hormone.  2,4-D is available as the free acid
but  is used, in agriculture and forestry, in formulations as a salt or
ester.

1.1.  Uptake, Accumulation, Elimination, and Biodegradation

    2,4-D does not persist in soil because of its rapid degradation.

    The  physico-chemical properties of 2,4-D acid and its formulations
have   an   important  effect   on   its  behaviour   in  environmental
compartments.

    The  bioavailability  to, and  uptake  by, aquatic  and terrestrial
organisms  is strongly  influenced by  the organic  matter  content  of
soils,  microbiological activity, and by  environmental conditions such
as  temperature and  pH.  Although  highly inconsistent,  the  data  on
dissipation and bioavailability in various soils demonstrate  a  marked
influence  of differences in the texture and mineral composition of the
soil.   In aerobic soils, with a high content of  organic material, and
at high pH values and temperatures, toxic effects are  limited  because
of rapid degradation of 2,4-D.

    Uptake  is followed by rapid excretion in most organisms.  With the
exception  of some algae,  the retention of  2,4-D by organisms  in the
environment cannot be expected, because of its rapid degradation.

    Some  microorganisms are capable of  utilizing 2,4-D as their  sole
carbon source.  Repeated application to soil stimulates the  number  of
organisms capable of degrading the compound.

1.2.  Toxicity to Microorganisms

    In  general  2,4-D  is  relatively  non-toxic  to  water  and  soil
microorganisms  at recommended field  application rates.  No  effect of
2,4-D  was recorded on 17 genera of freshwater and two genera of marine
algae  at  concentrations up  to 222 mg/litre.   No effect of  2,4-D on
respiration  of either sandy  loam or clay  loam soils was  observed at
concentrations up to 200 mg/kg.

    N-fixation by aquatic algae is affected at high  concentrations  of
2,4-D  acid (400 mg/litre).   An effect of  2,4-D esters on  N-fixation
occurs  from a concentration of 36 mg/litre upwards.  N-fixing algae in
topsoils  appear to be more  vulnerable to 2,4-D acid  than other algal
species.   The Cyanobacteria (blue-green  algae) are important  as  the
major N2 source in tropical ponds and soils.

    In  the range of 25.2 to 50.4 mg/litre, 2,4-D was inhibitory to all
types of soil fungi.

    Cell division was reduced in a green alga by 2,4-D at  20  mg/litre
and  stopped  at  50 mg/litre.   No  effect  was observed  on a natural
phytoplankton   community  after  exposure  to  2,4-D  at  1  mg/litre.

However,  exposure to  esters of  2,4-D reduced  productivity in  these
organisms.

1.3.  Toxicity to Aquatic Organisms

    At  recommended application rates,  the concentration of  2,4-D  in
water  has  been  estimated to  be  a  maximum of  50  mg/litre.   Most
applications  would lead to water  concentrations much lower than  this
(between 0.1 and 1.0 mg/litre).

    The  short-term toxicity data  on the effects  of 2,4-D free  acid,
its  salts, and esters  on aquatic invertebrates  is extensive.   Ester
formulations  are more toxic than the free acids or salts.  Sensitivity
variations  exist among species  in response to  the same  formulation.
Organisms  become more sensitive  to 2,4-D when  the water  temperature
increases.   Reproductive  impairment occurred  at concentrations below
0.1 of the short-term toxic levels determined for these formulations.

    LC50 values   for fish vary considerably.  This variation is partly
due  to differences in species sensitivity, chemical structure (esters,
salts, or free acid), and formulation of the herbicide.

    Although  the free acid  is the physiologically  toxic entity,  the
ester  formulations represent a major hazard to fish when used directly
as aquatic herbicides (because they are more readily taken up by fish).
Amine  salt formulations used  to control aquatic  weeds do not  affect
adult fish.

    The no-observed-effect-level (NOEL) varies with the species and the
formulation: less than 1 mg/litre (coho salmon) to 50 mg/litre (rainbow
trout).

    Fish larvae are the most sensitive life stage but are  unlikely  to
be affected under normal usage of the herbicide.

    Long-term   adverse   effects  on   fish   are  observed   only  at
concentrations  higher than those produced after 2,4-D has been applied
at recommended rates.

    Few  studies are related to the effects of environmental variables,
such  as  temperature and  water hardness, on  2,4-D toxicity to  fish.
Higher  temperature  possibly increases  the  toxicity.  This  might be
considered when assessing the safety of 2,4-D to fish during control of
aquatic weeds.

    Fish  detect and  avoid 2,4-D  only at  higher concentrations  than
those obtained under normal conditions of use.

    Amphibian  larvae are generally tolerant  to amine salts of  2,4-D;
the 96-h LC50 values  exceed 100 mg/litre.  Of the species tested, only
one  was  sensitive.   No  information  is  available  on  reproductive
development and differentiation or on tissue levels.

1.4.  Toxicity to Terrestrial Organisms

    Based  on the widespread use of 2,4-D and its formulations, insects
of many kinds could be exposed to the material.  Although the compounds
are generally classified as non-toxic for beneficial insects,  such  as
honey bees and natural enemies of pests, some adverse effects have been
reported on the early life-stages and adults of some insects.

    Esters are less toxic to insects than are salts or the free acid.

    Birds, and particularly the eggs of ground-nesting  species,  would
be  exposed to 2,4-D after spraying.  Food items could also be expected
to  be contaminated by the  herbicide.  However, most studies  on birds
and their eggs have been conducted at exposures far higher  than  could
be expected in the field.

    LD50 values   from acute oral  and from short-term  dietary  dosing
indicate  low toxicity  of 2,4-D  to birds.   In  longer-term  studies,
effects  have  only  been reported  at  extremely  high exposures  (for
example,   kidney  effects  after   dosing  in  drinking   water   with
concentrations  in excess of  the solubility of  the material).   There
have  been no  reported effects  on reproductive  parameters,  even  at
excessive exposure levels.

    A  single study reported adverse  effects on the embryos  of birds'
eggs  sprayed with 2,4-D.  Many  studies since have shown  no effect on
hatchability  of eggs and  no increased incidence  of abnormalities  in
chicks  even after very high exposure to 2,4-D.  Other work indicates a
very poor penetration of the eggshell by the herbicide.  It can only be
concluded  that after normal, or even after excessive, 2,4-D use, there
would be no effect on birds' eggs.

    Based  on the available data,  no generalization can be  made about
the  hazard  of  2,4-D to mammals in the field.  Data on voles indicate
that the herbicide poses no hazard.

1.5.  Effects of 2,4-D in the Field

    No  direct toxic effects, acute or long-term, of 2,4-D applications
under field conditions on any animals species have been  observed  thus
far.

    There are, inevitably, indirect effects resulting from the intended
selective herbicidal properties of the compound.  These  effects  would
result  from  the use  of any herbicide  or from other  methods of land
management.   There will, therefore, be effects for mammals, birds, and
insects   because  of  food   deprivation,  modification  of   habitat,
requirements for nesting, shelter, etc.

    The  application  of  2,4-D appears  to  be  less hazardous  to the
beneficial epigeal arthropod community than physical cultivation.

2.  PHYSICAL AND CHEMICAL PROPERTIES
               
    Details   of  the  physical   and  chemical  properties   of   2,4-
dichloropheoxyacetic  acid  (2,4-D)  are given  in Environmental Health
Criteria  29: 2,4-D (WHO,  1984).  The relevant  chapter is  summarized
here.

    The   structures  of  2,4-D   and  of  chemically-related   phenoxy
herbicides in common use are given in Fig. 1.  2,4-D is  a  chlorinated
form of a natural plant hormone (auxin).

    Some physical properties of 2,4-D and of the 2,4-D derivatives that
are used in agriculture are summarized in Tables 1 & 2.

    2,4-D  has growth-regulating and  herbicidal  properties in  broad-
leaved  plants.  Because of its solubility, 2,4-D is rarely used in the
form  of the acid; commercial  2,4-D herbicide formulations consist  of
the  more soluble forms such  as alkali salts, amine  salts, or esters.
These  are combined  with solvents,  carriers, or  surfactants and  are
marketed  in the form of  dusts, granules, emulsions, or  oil and water
solutions in a wide range of concentrations.

Table 1.  Physical properties of 2,4-D
-------------------------------------------------------
Molecular formula                   C8H6Cl2O3

Relative molecular mass             221.0

Melting point                       140 - 141 °C

Solubility in water                 slightly soluble

Solubility in organic solvents      soluble

Vapour pressure                     52.3 Pa at 160 °C

pKa at 25 °C                        2.64 - 3.31
-------------------------------------------------------

2.1.  Synthesis of 2,4-D

    2,4-D  is commonly prepared  by the condensation  of  2,4-dichloro-
phenol  with monochloroacetic  acid in  a strongly  alkaline medium  at
moderate temperatures or by the chlorination of phenoxyacetic acid, but
this  method leads to  a product with  a high content  of 2,4-dichloro-
phenol   and  other  impurities.   Higher  reaction  temperatures   and
alkaline  conditions  during  the  manufacture  of  2,4-D  increase the
formation of polychlorinated dibenzo- p -dioxin   (CDD) by-products.  One
formulation  of  2,4-D  was found  to  contain  6.8 µg/kg  of  2,3,7,8-
tetrachlorinated  dibenzo- para -dioxin    (Hagenmaier, 1986).   In other
amine  and  ester  formulations,  levels  of  this  dioxin  were   non-
detectable,  i.e., < 1 µg/kg  (WHO, 1984).   The alkali metal salts  of
2,4-D  are produced by the reaction of 2,4-D with the appropriate metal
base.  Amine salts are obtained by reacting  stoichiometric  quantities
of  amine  and 2,4-D  in a compatible   solvent.  Esters are  formed by
acid-catalysed  esterification with azeotropic distillation of water or

by  direct synthesis in which the appropriate ester of monochloroacetic
acid is reacted with dichlorophenol to form the 2,4-D ester.

FIGURE 1

2.2.  Important Chemical Reactions of 2,4-D

    Pyrolysis   converts   various  amine   salts   of  2,4-D   to  the
corresponding amides.  Pyrolysis of 2,4-D and its derivatives is likely
to produce certain CDD isomers.  2,4-D is readily photodegraded.

2.3.  Volatility of 2,4-D Derivatives

    2,4-D esters with short-chain alcohols are highly  volatile.   This
influences  the  effectiveness of  their  application to  target crops,
their effects on neighbouring crops, and the degree of contamination of
the  atmosphere.   2,4-D  alkali salts  or  amine  salts are  much less
volatile than esters, and these products are to be preferred  when  the
use  of 2,4-D esters might lead to evaporative 2,4-D losses and to crop
damage or damage to the surrounding environment.

    Details  of  technical  compositions,  impurities,  and  analytical
methods  can  be  found  in  Environmental  Health  Criteria  29:  2,4-
Dichlorophenoxyacetic acid (WHO, 1984).

Table 2.  Vapour pressure and solubility of 2,4-D salts and esters
--------------------------------------------------------------------------------
Compound                  Vapour pressurea        Solubility
--------------------------------------------------------------------------------
2,4-D free acid           0.4 mmHg (160 °C)       0.09% in water (25 °C),
                                                  85% in acetone (25 °C)

dimethylamine salt                                300% in water (20 °C),
                                                  soluble in acetone

isopropyl ester           1.4 x 10-3 mmHgb        insoluble in water, soluble
                          4.6 x 10-5 mmHgb        in most organic solvents

butoxyethanol ester       4.5 x 10-6 mmHgb        insoluble in water, soluble
  (butylethyl ester)                              in most organic solvents

ethylhexyl ester          2.0 x 10-6 mmHgb        insoluble in water, soluble
                                                  in organic solvents

isooctyl ester            2.0 x 10-6 mmHgb        insoluble in water, soluble
                                                  in organic solvents

propyleneglycol butyl     3.0 x 10-6 mmHgb        insoluble in water, soluble
  ether ester                                     in organic solvents

methyl ester              2.3 x 10-3 mmHgb

ethyl ester               1.1 x 10-3 mmHgb

butyl ester              3.97 x 10-4 mmHgb
--------------------------------------------------------------------------------
a  1 mmHg = 0.133 kPa.
b  Vapour pressures of esters were determined at high temperatures by gas-
   liquid chromatography, and these values are the result of extrapolation
   to 25 °C.  Values vary considerably between authors as a result of this
   extrapolation; original values at high temperatures agree.  Results are
   presented here as an indication of relative vapour pressure at working
   temperature.  Values from Flint et al. (1968) and Jensen & Schall
   (1966).

3.  SOURCES OF ENVIRONMENTAL POLLUTION

    The following is a summary of the chapter from Environmental Health
Criteria 29: 2,4-Dichlorophenoxyacetic acid (WHO, 1984).

3.1.  Production of 2,4-D Herbicides

    Comprehensive  statistics on 2,4-D herbicide production or use were
not   available   for  review.   According  to  the  US  Department  of
Agriculture,  3 x 108 kg  of  total herbicides  were used  in  the  USA
alone,  in  1981.  In  the past, 10%  of the herbicide  used was 2,4-D,
which would account for a total use in the USA of about  3 x 107    kg.
In  1975, an estimated 5 x 106 kg  were produced in the United Kingdom.
World-wide  use  of herbicides  and  annual production,  which probably
exceeds 5 x 107 kg/year, are increasing.

3.2.  Uses

    2,4-D  alkali  or amine  salts or esters  are used as  agricultural
herbicides  against broad-leaved weeds in  cereal crops, as well  as on
pastures  and  lawns,  in parks, and on golf courses, at rates of about
0.2  to 2.0 kg active ingredient (acid equivalent) per hectare.  Esters
are also used at rates of up to 6.0 kg (acid equivalent) per hectare to
suppress weeds, brush, and deciduous trees along rights-of-way  and  in
conifer plantations and conifer reafforestation areas.

    Granular formulations of 2,4-D are used as aquatic herbicides in or
along irrigation and other canals, in ponds, and lakes at rates ranging
from 1 to 122 kg/ha.

    2,4-D   products  can  be used  at very  low application  rates  as
growth regulators by application of aqueous foliar sprays containing 20
to  40 mg 2,4-D/litre on apple trees to reduce premature fruit-drop, on
potato  plants to increase the  proportion of medium-size tubers  or to
intensify  the tuber skin  colour of the  red varieties, and  in citrus
culture  to reduce pre-harvest fruit-drop and to increase fruit storage
life.

    The  highly volatile ethyl, isopropyl,  and butyl esters are  being
replaced by low-volatile esters or by amine salts to reduce crop damage
resulting   from  2,4-D  vapour  drift,  and  to  decrease  atmospheric
pollution.

    During  recent  years,  the use  of  2,4-D  and 2,4,5-T  in  parks,
forested recreation, and other areas frequently used by the public, has
been  reduced in  some countries  because of  increasing concern  about
possible toxic effects, especially in relation to CDDs.

3.3.  Disposal of Wastes

    Environmental  pollution with 2,4-D  may occur as  a result of  the
production  and  disposal  of 2,4-D,  or  of  its by-products,  and  of
industrial  effluents.  Such pollution  will be generally  localized to
the  production site and to areas of waste dumping, and it is likely to
be  more dispersed  if disposal  or leaching  has occurred  into  water
courses.    Disposal of  unused 2,4-D  in agriculture  and  washing  of
equipment  may result in localized land pollution and also pollution of
water supplies through direct contamination or leaching from soil.

4.  UPTAKE, ACCUMULATION, ELIMINATION, AND BIODEGRADATION

 Appraisal

     2,4-D does not persist in soil because of its rapid degradation.

     The  physico-chemical properties of 2,4-D acid and its formulations
 have   an   important  effect   on   its  behaviour   in  environmental
 compartments.

     The  bioavailability  to, and  uptake  by, aquatic  and terrestrial
 organisms  is strongly  influenced by  the organic  matter  content  of
 soils,  microbiological activity, and by  environmental conditions such
 as  temperature and  pH.  Although  highly inconsistent,  the  data  on
 dissipation  and  bioavailability in various soils demonstrate a marked
 influence  of differences in the texture and mineral composition of the
 soil  (Graham-Bryce, 1972).  In aerobic  soils, with a high  content of
 organic material, and at high pH values and temperatures, toxic effects
 are limited because of rapid degradation of 2,4-D.

     Uptake  is followed by rapid excretion in most organisms.  With the
 exception  of some algae,  the retention of  2,4-D by organisms  in the
 environment cannot be expected, because of its rapid degradation.

     Some  microorganisms are capable of  utilizing 2,4-D as their  sole
 carbon source.  Repeated application to soil stimulates the  number  of
 organisms capable of degrading the compound.

4.1.  Biodegradation

    2,4-D  is  readily  and rapidly  degraded  in  soil.   Warm,  moist
conditions  and  addition  of  organic  matter  stimulate  degradation.
Autoclaving  the  soil  and  inhibiting  bacterial  metabolism   reduce
degradation.    The  kinetics  of  2,4-D   disappearance  suggest  that
microorganisms  are responsible.  Particular species of microorganisms,
of various types, have been isolated and shown to degrade phenoxyacetic
acid  herbicides  in pure  culture.   Degradation of  the phenoxyacetic
acids  proceeds by two main  pathways.  These are via  a hydroxyphenoxy
acetic   acid  intermediate  or  via  the  corresponding  phenol.   The
literature has been reviewed by the two workers principally responsible
for this evidence (Audus, 1960, 1964; Loos, 1969).  Some microorganisms
are  capable of using 2,4-D  as their sole carbon  source.  More often,
2,4-D  is co-metabolized with another carbon source.  Regular treatment
of  soil  with  2,4-D stimulates  the  numbers  of organisms  which are
capable  of  degrading  the  compound.   Treatment  with  other phenoxy
herbicides  can  also  lead to  an  increase  in organisms  capable  of
degrading 2,4-D.

    Butler  et  al.  (1975a) exposed  21  species  of freshwater  algae
isolated  from natural lake  water to 2,4-D  butoxyethanol ester, at  a
concentration of 0.01 mg/litre, and looked for degrading ability.  Most
of  the cultures fully degraded 2,4-D within 2 weeks.  A single culture
retained  64% of the added 2,4-D, while seven isolates reduced 2,4-D to
less than 20% of the amount added.  The remaining isolates showed 2,4-D
recoveries ranging from 22% to 53%.

    Le   Van  To  (1984)   isolated  six  species   of   microorganisms
from   soil   previously   treated   with   herbicides.    These   were
 Flavobacterium    peregrinum,  Pseudomonas   fluorescens,  Arthrobacter
 globiformis,   Brevibacterium sp., Streptomyces viridochromogenes,  and
an   unidentified  Streptomyces species.  Flavobacterium was   the  most
active   organism  in  degrading   2,4-D;  degradation of  20 mg/kg  of
2,4-D  was  complete  after  20  to  30  days.   In  a  liquid  medium,
 Flavobacterium  degraded  93.5% of added  2,4-D within 80  h. The  time
required  to degrade half of the 2,4-D added to a sterilized soil along
with nutrient was estimated at 3 days.  Li-Tse Ou  (1984)  investigated
the  breakdown of  2,4-D in  two types  of soil  under  dry  and  moist
conditions   and   at   two   different   temperatures.    Numbers   of
microorganisms  degrading 2,4-D were also  estimated.  Generally, 2,4-D
disappeared more rapidly from moist soil;  after 14 days of a slow rate
of  disappearance,  however,  the removal  rate  from  dry, sandy  soil
increased.   Numbers of organisms  degrading 2,4-D were  initially much
lower  in sandy than in clay loams.  However, numbers increased rapidly
in  sandy  soils  after the addition of the herbicide and, as a result,
2,4-D was eventually degraded more rapidly in sandy than in clay loams.
In  moist conditions, at 25 °C,  the half-life of 2,4-D  was 7 days  or
less, whereas in dry conditions, at 35 °C, it could be as long  as  250
days.   These latter conditions are  unlikely to apply in  most natural
conditions where 2,4-D is likely to be used.

    Rosenberg  & Alexander (1980) incubated sewage-sludge bacteria with
2,4-D  and found that nearly all of the herbicide had disappeared after
7  days.   Subsequent  additions of  2,4-D  led  to destruction  of the
compound  without a lag period;  this suggests selection for  organisms
capable of degrading the compound.  Similar results were obtained using
bacteria  from soil.  The time  needed for the disappearance  of 90% of
the  added  2,4-D  was  14  days  with  soil  inocula.    2,4-D   added
subsequently was reduced by 70% within 3 to 4 days.   Various  tropical
soils  were  used  in the experiment and all showed a high capacity for
degrading  2,4-D.  Thompson et al. (1984) determined the persistence of
2,4-D applied at recommended rates in agricultural soils in Canada.  In
all but one soil, a sandy loam, the concentration had declined  by  50%
within  7 days.  Sattar &  Paasivirta (1980) showed slower  degradation
of  2,4-D  in  acid  soils.  It took  6 weeks for  50% of the  2,4-D to
disappear  from the soil  and 7% was  still left after  24  weeks.   In
water-logged soil, there was reduced degradation of the herbicide.

    Lewis   et  al.  (1984)   studied  bacterial  breakdown   of  2,4-D
butoxyethyl ester and the effects of adding various extra components to
the medium.  The addition of unfiltered, spent fungal medium from which
the  majority of the fungus had settled out could be either stimulatory
or inhibitory to degradation rates of the herbicide; this  depended  on
the   particular  fungus  species  cultured  in  the  medium.   Further
investigation  showed that effects were primarily due to differences in
pH.   Reduction of the pH below 6 inhibited bacterial transformation of
the  compound.  Fungi commonly release  large amount of organic  acids.
The  addition of spent fungal  medium inhibited the breakdown  of 2,4-D
ester.   Buffering  the added  fungal  medium reduced  this  inhibitory
effect;   indeed,  some stimulation  of  breakdown occurred  after  the
addition  of buffered,  spent medium.   The addition  of nutrients,  or
other   bacteria  which  did   not  transform  2,4-D,   stimulated  the
transformation  of the herbicide.  The  authors consider that the  most

likely   explanation  for  this   phenomenon  is  induction   of  other
transforming enzymes.  With increasing substrate concentration, further
enzyme  systems  are  induced  in  bacteria.   The  presence  of  other
organisms may stimulate the induction of these other enzymes  at  lower
substrate  concentrations than would  normally induce them.   Increased
biomass of transforming bacteria in the presence of competing organisms
contributes  to  increased transformation  rates.   The nature  of  the
microbial  community  can, therefore,  greatly  change the  ability  of
degrading bacteria to transform 2,4-D and other xenobiotics.

    O'Connor  et al. (1981) found that 2,4-D applied at about 1.5 mg/kg
was  readily degraded  in soil.   Adding extra  carbon in  the form  of
dried,  digested sewage  sludge had  a short-term  effect in  enhancing
degradation of the compound.  Torstensson (1975) measured the half-life
of  2,4-D degradation in cultures  of soil microorganisms at  different
pH.   In the pH  range of 8.5  to 5.0, the  half-life  changed  little,
ranging  from 5 to 8  days.  At pH 4.5,  the half-life increased to  21
days and, at pH 4.0, increased further to 41 days.

    Lieberman  & Alexander (1981) added  2,4-D to inocula of  municipal
sewage and monitored the biological oxygen depletion (BOD) as a measure
of  degradation.  The herbicide  was added to  carbon-depleted  inocula
such that the 2,4-D represented the sole carbon source.  Less  than  5%
of the available oxygen was depleted, indicating poor biodegradation of
2,4-D  because  of low  numbers of organisms  capable of degrading  the
herbicide  as their sole carbon  source.  A separate study  showed that
2,4-D was not toxic to microorganisms in sewage.

    Fournier  (1980) showed that,  while 2,4-D treatment  increased the
numbers  of soil microorganisms  capable of metabolizing  2,4-D as  the
sole  carbon source and those capable of co-metabolizing the herbicide,
this  increase was dependent  on the concentration  of 2,4-D used.   At
concentrations  of  2,4-D  between  5  and  50  mg/litre, there  was  a
significant  increase in the  numbers of organisms  metabolizing 2,4-D,
and at 5 mg/litre there was a very pronounced increase in organisms co-
metabolizing the compound.  At much higher (500 mg/litre) or much lower
(1.2 µg/litre)   2,4-D  concentrations, there  was  no increase  in the
numbers of either metabolizing or co-metabolizing organisms.

    Sandmann  &  Loos (1984)  estimated  the numbers  of microorganisms
capable of degrading 2,4-D in soils with and without  the  `rhizosphere
effect'  of two plants, African clover  (Trifolium africanum)  and sugar
cane  (Saccharum   officinarum).     The   `rhizosphere  effect'   is  a
phenomenon  which occurs in close association with the roots of plants,
where  material  from the  root or the  metabolic activity of  the root
tissue  affects  the  surrounding soil.   Particularly high, stimulated
populations  were associated with sugar cane.  A similar effect, but to
a  lesser degree, was found with clover.  In the three sugar cane soils
examined,  and their corresponding  controls, the numbers  of organisms
were  46 400, 156 000, and 40 700 per g of soil, with rhizospheres, and
178,  1480, and 6170 per g of soil, without rhizospheres, respectively.
Seibert  et al. (1982)  failed to demonstrate  a rhizosphere effect  on
2,4-D  degradation in glasshouse studies  using soils with and  without
maize roots.

    Norris  & Greiner (1967) investigated  the degradation of 2,4-D  in
forest leaf litter.  Litter from either alder, ceanothus,  vine  maple,
bigleaf   maple  or Douglas  fir showed comparable  ability to  degrade
2,4-D, the recovery of 2,4-D being between 60% and 70% after 15 days of
incubation.   In a second series of experiments, different formulations
of  2,4-D were added to  alder litter.  About 50%  of the free acid  of
2,4-D  was  degraded  within 15  days.   Triethanolamine  salt and  two
commercial  formulations (`solubilized acid'  and isooctyl ester)  were
degraded  less  than  the pure  acid.   There  was between  30% and 40%
degradation of these preparations over 15 days.

    Nesbitt  & Watson (1980) related  the degradation rate of  2,4-D in
river  water  to  the nutrient  levels,  sediment  load, and  dissolved
organic  carbon content  of the  water.  The  addition of  sediment  or
inorganic  nutrients increased the  rate of 2,4-D  degradation, whereas
the addition of organisms capable of degrading 2,4-D did  not  increase
the  rate of breakdown of  the herbicide.  This finding  indicated that
the  limiting  factor  in breakdown  of  2,4-D  in river  water was not
numbers of organisms but the nutrient status of the river.  The authors
noted  that in winter, when  the river was in  peak flow and the  water
temperature  below  that  for optimum  microbial  activity, appreciable
amounts  of the herbicide would be washed into the estuary.  An earlier
pilot  study of  seasonal changes  in the  capacity of  river water  in
Western  Australia  to degrade  2,4-D  (Watson, 1977)  indicated  clear
seasonal   differences  in  both  river  water  concentrations  of  the
herbicide  and the degrading capacity  of river water.  Several  rivers
were   studied   and  differences   were  related  to   the  amount  of
agricultural  run-off, the sediment content  of the water, river  flow,
and  temperature.  Rivers receiving agricultural run-off degraded 2,4-D
better than those receiving run-off principally from forests.  This was
presumed  to be the result  of the preconditioning of  organisms to the
herbicide;  the  investigation corrected  for  nutrient content  of the
water which had been previously shown to affect degradation.

    Spain  &  Van  Veld  (1983)  looked  at  the degrading  ability  of
microbial  communities  taken  from  sediment  cores  from  freshwater,
estuarine,  and marine sites.   Some cores were  pre-exposed to  2,4-D.
Cores from freshwater sites showed increased degradation of 2,4-D after
pre-exposure  to the compound, whereas  estuarine and marine cores  did
not show this effect.  The adaptation of freshwater cores  was  maximal
after 2 weeks and no longer detectable 6 weeks after pre-exposure.

4.2.  Uptake and Accumulation by Organisms

 Appraisal

     Many studies on the accumulation of 2,4-D have  used  radioactively
 labelled  herbicide and have monitored uptake by simple counting of the
 label.   This fails to take into account that the label could have been
 removed  from the parent molecule  by metabolic breakdown.  Values  for
 uptake  should, therefore,  be treated  as a  maximum  possible  uptake
 value   for   2,4-D.   Such  data  would  not  normally  be  considered
 acceptable.   However, the accumulation of  2,4-D is so low  that these
 data serve to illustrate that little of the herbicide is accumulated.

4.2.1.  Laboratory studies

    Eliasson    (1973)  sprayed  leaves  of  3-year-old  aspen  (Populus
 tremens)  with  the  butoxyethanol  ester  of  2,4-D  at  0.5  kg  acid
equivalent/litre.   The  plants  were  then  kept  in   an   open-sided
glasshouse  and  residues  of  2,4-D  were   monitored.   Most  of  the
herbicide  remained in, or on, the sprayed leaves.  The average residue
level was 2300 mg/kg fresh weight 1 day after spraying.  This level had
fallen to 1300 mg/kg after 37 days and, by day 365, the average residue
level  was  870  mg/kg.  This  was  a  very high  application  rate and
indicates that there is no foliar uptake of 2,4-D by plants.

    Glynn  et al. (1984) exposed coral  Pocillopora damicornis  to three
concentrations   of  2,4-D  sodium   or  amine  salts  at 0.1, 1.0,  or
10.0  mg/litre.  The  maximum concentration  of 2,4-D  found  in  coral
tissue was 0.137 mg/kg after exposure to the amine salt at 10 mg/litre,
but residues were not related to the 2,4-D exposure concentration.  The
highest  bioconcentration factor (BCF) of 1.33 was found after exposure
to 0.1 mg/litre of the amine salt of 2,4-D, i.e., the  coral  contained
1.33 times the concentration of 2,4-D in water.

    Metcalf  &  Sanborn   (1975) introduced  14C-labelled    2,4-D into
model  ecosystems  consisting  of an alga  Oedogonium,  an aquatic plant
 Elodea,  a   snail  Physa,   and  the  mosquito  fish  Gambusia.    Total
14C   in the water was equivalent to 0.205 mg 2,4-D/litre.  The highest
BCF  was  in the  alga (26.8, based  on measurement of  radioactivity).
Analysis  of all components of the ecosystem for 2,4-D, rather than the
radiolabel,  revealed none of the parent compound.  The BCF, therefore,
refers  to breakdown products  rather than 2,4-D  itself.  Gile  (1983)
introduced 14C-labelled   2,4-D, as the butyl ester, into  a  simulated
ryegrass  ecosystem.  The system consisted of a sandy loam soil, annual
ryegrass,  several  invertebrates,  and grey-tailed  voles.  Voles were
introduced  10  days  after spraying  2,4-D  as  a foliar  spray at the
equivalent  of 1 kg/ha.  The  experiment was terminated after  1 month.
Plant material contained an average of 8.9 mg/kg; this  was  identified
as  being  mostly  2,5-dichloro-4-hydroxyphenoxyacetic  acid.   Residue
levels  in animals (based on  unidentified 14C   residues) ranged  from
0.31 mg/kg in snails to 5.28 mg/kg in pillbugs (isopods).

    Freitag et al.(1982) measured the bioaccumulation of 14C-2,4-D   in
an alga  Chlorella fusca  and a fish, the golden orfe. They  measured  a
24-h  static  BCF  of 6 for the alga, and a 3-day static BCF of <10 for
the  fish.  This measurement was based on radioactivity and, therefore,
did  not distinguish  between the  parent compound  and  its  breakdown
products.

    Schultz  (1973) examined uptake  and loss of  14C-2,4-D   dimethyl-
amine  salt  by  organs  of  three  species  of fish  (channel catfish,
bluegill   sunfish,  and  largemouth  bass),   exposed to 0.5, 1.0,  or
2.0  mg/litre of 2,4-D acid equivalent.  After exposure to the  highest
concentration  of  2,4-D  dimethylamine  salt,  there  was   detectable
radioactivity in all organs examined.  Bile showed the highest residues
of 14C   in all three species after 1 week.  For the remainder  of  the
exposure  period of 12 weeks, there was an increase of radioactivity in
other  organs  and  a decrease in the bile.  At the end of the exposure
period,  there  was  no clear  pattern  to  residue levels  of 14C   in

different   organs.   These levels  ranged from 5.04  mg/kg in bile  to
35.5 mg/kg in posterior kidney for the channel catfish.  For largemouth
bass, the range was from 1.32 mg/kg in muscle to 7.29 mg/kg  in  liver.
For  the sunfish, the lowest  residue was 24.75 mg/kg  in bile and  the
highest  322.7 mg/kg in the  pyloric caeca of the  gut.  After 84  days
exposure  to the dimethylamine salt  at 2 mg/litre, levels  of 14C   in
the  muscle  of catfish,  bass, and sunfish  were equivalent to  0.953,
0.035,  and 1.065 mg  2,4-D/kg, respectively.  No   analysis for  2,4-D
itself  was carried out.  A second study exposed the three fish species
for  2 weeks to 14C-2,4-D    dimethylamine salt at 1  mg/litre and then
for  a further 4 weeks to clean water.  The disappearance of  14C   was
measured.   Loss of 14C   was slow at first but by 4 weeks most tissues
had shown a decline in residues.  Samples were analysed for  2,4-D  but
none  was  detectable,  suggesting  that  the  14C    measured  was  in
breakdown  products.  The values for  2,4-D residues in this  and other
studies using 14C-labelled   material should, therefore, be regarded as
overestimates of retained 2,4-D.  Uptake of 14C-2,4-D   was examined at
two  different temperatures, 17 °C and 25 °C.  The highest residues  of
14C    detected  in  fish  were equivalent to 0.122 mg 2,4-D/kg, but no
2,4-D  could be found after analysis, except in  bluegill sunfish after
14  days.   Loss  of 2,4-D  did  not,  therefore, seem  to  change with
differing  temperature  over  this range.   A  similar   study, at  two
different  water pH values, showed  significantly more 14C   uptake  in
all  three species at the more acidic pH.  Analysis of fish tissues for
2,4-D  by  gas-liquid  chromatography showed  non-detectable, or trace,
levels in most samples.  Only in bluegill sunfish after 7 and  14  days
were  residues measurable.  These  2,4-D residues showed  the  opposite
trend to the 14C   results; there was more 2,4-D in fish exposed at the
more alkaline pH.  The authors suggest that metabolism of the herbicide
in the fish is suppressed at alkaline pH.

    Sigmon (1979) exposed bluegill sunfish to 2,4-D butyl  ethyl  ester
(3  mg/litre) at three different  temperatures, 20, 25, and  30 °C, and
measured  the tissue content of 2,4-D after 8 days.  None of the groups
differed from the controls, residues being <0.05 mg/kg.

    Bluegill  sunfish  and  channel  catfish   took  up  <0.5%  of  the
available  14C   when exposed  to 14C-2,4-D   dimethylamine  salt at  2
mg/litre  (with 1 litre of  water per fish) for  7 days (Sikka et  al.,
1977).    A   maximum   total  14C     concentration  in  the fish  was
reached  after 24  h and  did not  change significantly  over 14  days.
Bluegill sunfish attained a total body concentration of 0.9  mg/kg  and
catfish  0.2  mg/kg at  24 h.  These  values were 2,4-D  equivalents of
14C   measured;  the compound was not analyzed directly.  When bluegill
sunfish  were injected intraperitoneally with 14C-2,4-D   dimethylamine
salt,   at   dose   levels  of   1   or   2.5 mg/kg  body  weight, they
excreted  90%  of   the dose  within 6  h of  treatment.  In  a similar
experiment,  Stalling  & Huckins   (1978)  exposed bluegill  sunfish to
14C-2,4-D     dimethylamine   salt  at  2  mg/litre  and  measured both
14C    and 2,4-D in fish and water samples over the following 12 weeks.
Radioactivity   was  detected  in   tissues  and  increased   over  the
experimental period, but there was no measurable 2,4-D;  the  detection
limit  of  the  method was  0.1  mg/kg.   An  in  vivo   intraperitoneal
injection of 110 µg of 14C-2,4-D   was followed by rapid elimination.

    Rodgers  & Stalling (1972)  measured uptake of  14C-2,4-D   butoxy-
ethanol  ester by three species  of fish, which were  exposed to either
0.3  or  1.0  mg/litre and sampled over the next 168 h.  Some fish were
fed  and  some  fasted.  Radioactivity  in  a  variety of  tissues  was
determined; the maximum levels were found within 3 h of exposure in fed
fish.   After this, levels declined over the remaining sampling period,
and  by the end of  the experiment, residues were  negligible.  The one
exception was the gall bladder, which consistently contained more 2,4-D
than other tissues.  Results were different for fasted fish.  In almost
all  organs  of fasted  fish, uptake of  2,4-D was slower  than for fed
fish,  although the levels  reached were eventually  two to five  times
higher than in fed fish.  Analysis of the residues showed that only the
liver  ever contained the  herbicide in the  ester form.  In  all other
tissues, only the acid was present.

    Shcherbakov  & Poluboyarinova (1973) monitored  the accumulation of
2,4-D in  carp  and  Daphnia.   The 2,4-D was added as the  butyl  ester
at  concentrations ranging from  0.006 to 5  mg/litre; the  recommended
usage   rate  for  this  ester  leads to water concentrations  of about
0.5  mg/litre.  Analyses of fish  tissues were made for  both the ester
and  the  acid.   The highest BCF for the ester, at 395, was found with
fish  after a 7-day  exposure to 0.5  mg/litre.  Acid accumulation  was
lower than that of the ester.  The experiment lasted for 70  days.   At
day  10 and after,  only trace amounts  of ester were  found  in  fish.
Small  amounts  of  2,4-D acid  were found  at day  10, but  only trace
amounts after day 70.  Residues of 2,4-D ester in  Daphnia  varied  from
23.9 to 518 mg/kg, according to the exposure concentration.

    Two experiments have been carried out on the grey  slug    Derocerus
 reticulatum  by Haque & Ebing  (1983) using 14C-labelled    2,4-D acid.
The  first study, a contact  experiment, exposed the slugs  to 2,4-D in
contaminated  soil at 1.1  mg/kg.  The body  content of 2,4-D  in slugs
reached  equilibrium (0.014 mg/kg)  after 15 days;  this represented  a
BCF  of  0.013  based on  radioactivity.   In  the  second  experiment,
slugs were exposed via the food using carrot discs containing 1.1 mg/kg
slug body weight per day over 5 days.  Residues of 14C   in  the  slugs
increased  during the feeding period, peaking at 5.5 mg/kg.  During the
following  7  days,  residues were  monitored  to  investigate loss  of
radioactive  material.   At  the end  of  the  experiment, on  day  12,
residues  were comparable to  those at the  end of the  feeding period.
During  the course of feeding 2,4-D-contaminated carrots, more than 80%
of  the ingested dose of  radioactivity was excreted rapidly;  only 20%
was retained.  There was no attempt to characterize the 14C   residues;
these  may,  therefore,  represent  either  2,4-D  or   its   breakdown
products.

    Chickens   given  a  single   oral dose of  100, 200, or  300 mg/kg
body   weight  reached maximum plasma  levels of 2,4-D of  90, 130, and
250 µg/ml,  respectively.  Plasma  levels in all groups had  fallen  to
15 µg/ml    or  less  after 24  h.  Continuous  dosing  of  chickens at
300 mg/kg per day led to a faster rate of elimination of the daily dose
of 2,4-D with time (Bjorklund & Erne, 1966).

4.2.2.  Field studies

    Cope  et al. (1970) treated experimental ponds with 2,4-D propylene
glycol  butyl  ether  ester to  give  water  concentrations up  to  and
including 10 mg/litre.  No detectable 2,4-D was found in  fish  exposed
to  1  mg/litre or  less of the  herbicide, but residues  were found in
bluegill  sunfish  exposed to  5 or 10  mg/litre.  The highest  residue
(2 mg/kg)  was found  1 day  after application.   Residues  were  still
detectable    after   3   days  but   not   subsequently.    Vegetation
 (Potamogeton  nodosus)  and   bottom   sediment  contained  residues of
50.0 and 3.0 mg/kg, respectively, 2 days after treatment with the 2,4-D
ester  at 10 mg/litre.  The herbicide was still detectable at 0.1 mg/kg
in  sediment  after  44 days  but  not  thereafter.  At  44  days after
treatment, there were residues in the plant of 1.2 mg/kg;  this  amount
declined to 0.1 mg/kg after 94 days.

    Following  the field application  of 2,4-D butoxyethanol  ester  at
22.5 kg/ha, Whitney et al. (1973) measured residues of the herbicide in
fish, crustacea, and insect larvae over a 3-week period.  The herbicide
had  been applied to  control eurasian water  milfoil.  Some 2,4-D  was
taken  up by these various  species; the highest residue  concentration
was  0.24  mg/kg  in largemouth  bass  after  8 days.   All residues in
organisms  were  below  0.1 mg/kg  after  3  weeks.  No  2,4-D could be
detected  in water in 33  samples taken after treatment,  the detection
limit being 0.10 mg/litre.  The highest reported concentration of 2,4-D
in mud was 0.65 mg/kg, 10 days after treatment, but in most samples the
herbicide  level  in  mud  was  much  lower  and  in  several  it   was
undetectable.

    Hoeppel  &  Westerdahl (1983)  treated four areas  (10 ha each)  of
dense water milfoil beds in Lake Seminole, Georgia, with  either  2,4-D
dimethylamine  salt  or  2,4-D butoxyethanol  ester,  at  each  of  two
application rates (22.5 or 45 kg/ha).  Both formulations were converted
to  2,4-D free acid within 24 h.  Maximum water concentrations achieved
in  the  high  rate (45 kg/ha) areas were 3.6 and 0.68 mg/litre for the
dimethylamine salt and butoxyethanol ester, respectively.  There was no
detectable  uptake of 2,4-D into  fish in those areas  treated with the
dimethylamine  salt.  In the ester-treated areas, 4 out of 24 game fish
sampled  contained low levels of  2,4-D in muscle (the  highest residue
being  0.29 mg/kg) and 18  out of 20 gizzard  shad contained detectable
2,4-D in muscle (the highest residue being 6.9 mg/kg).  No fish sampled
more than 13 days after treatment contained detectable 2,4-D.

    Schultz  & Harman (1974) treated nine experimental ponds with 2,4-D
dimethylamine salt at three concentrations: 2.24, 4.48, and 8.96 kg/ha.
Samples of water, bottom sediment, and fish were taken over  147  days.
Maximum  water and sediment concentrations of 2,4-D were 0.692 mg/litre
and  0.17  mg/kg,  respectively.  Of  307  fish  sampled, 45  contained
detectable  residues of 2,4-D.  The  highest residue measured was  in a
channel  catfish at 1.075 mg/kg 1 day after treatment.  All residues in
fish after 28 days were less than 0.005 mg/kg; most were undetectable.

    Smith  & Isom (1967) measured  uptake and retention of  2,4-D after
treatment  of two  field sites  for control  of watermilfoil  with  the
butoxyethanol  ester.   The  first site  was  treated  with a  granular
formulation  at a rate of  112 kg/ha.  One bluegill  sunfish    (Lepomis
 macrochirus)  contained 0.15 mg 2,4-D/kg on day 50 after treatment. All

other  fish,  sampled  between  72  h  and  50  days  after  treatment,
contained  less than 0.14 mg/kg, which was the limit of detection.  Two
samples  of several species of mussel, held in cages for 96 h following
spraying, showed residues of 0.38 and 0.7 mg/kg.  Water levels of 2,4-D
reached  a peak of 37 mg/litre within 1 h of application and had fallen
to  less  than  1 µg/litre  within  8  h.   Mud samples  contained very
variable levels of 2,4-D residues, ranging between 0.14 and 58.8 mg/kg.
The  highest residue was found 10 months after application.  The second
site  was treated at  the lower rate  of 45 kg/ha.   All  fish  sampled
between  15 days and 9  months after 2,4-D application  showed residues
of less than 0.14 mg/kg.  Mussels sampled between 1 and 42  days  after
application  contained residues ranging  between <0.14 and  1.12 mg/kg.
Water  levels peaked  at 157 µg/litre,   1 h  after spraying,  and  mud
residues ranged from <0.14 to 33.6 mg/kg.

    Coakley  et al. (1964) measured residues in organisms at the center
of a 0.4-ha field plot sprayed with 2,4-D butoxyethanol ester at a rate
of  33.7 kg/ha for watermilfoil  control.  Two days after  application,
oysters  (Crassostrea  virginica),  clams  (Mya arenaria),  fish  (Lepomis
 gibbus),  and blue crabs  (Callinectes sapidus)  contained 3.5, 3.7, 0.3,
and <0.8 mg/kg, respectively.

    In  1971,  over  2800  ha in Loxahatchee  National Wildlife  Refuge
were  sprayed with the   dodecyl-tetradecyl amine salts  of 2,4-D at  a
rate of 4.48 kg/ha.  The initial application of 2,4-D was  followed  by
spot treatments of the same formulation and/or the  dimethylamine  salt
of  2,4-D.  The highest water  concentration (0.037 mg/litre of  2,4-D)
was  measured  1 day after the initial application.  Of 60 fish sampled
in  the  area, 19  had measurable residues  of 2,4-D but  only three of
these  were greater than  0.1 mg/kg; the  highest recorded residue  was
0.162  mg/kg.  Breast muscle  and liver of  a bird, the  common Florida
gallinule  Gallinula  chloropus,  had residues  of 0.3  and 0.675 mg/kg,
respectively, 1 day after spraying.  No residues were found in the bird
4 days after spraying (Schultz & Whitney, 1974).

    Plumb   et  al.  (1977)  treated   sprouting  chamise    (Adenostoma
 fasciculatum)  with the polyethylene glycol butyl ether ester of  2,4-D
at  a rate of  3.4 kg acid  equivalent/ha.  A maximum  concentration of
herbicide  (95.2  mg/kg)  was found  in  the  plant within  15  min  of
application.   A  residue of  3.8 mg 2,4-D/kg  remained in, or  on, the
plants  (shoots  which  had  been  originally  sprayed)  1  year  after
treatment.  When Radosevich & Winterlin (1977) applied the butoxypropyl
ester   of 2,4-D  to a  chaparral area  at a  rate of  4.5  kg/ha,  the
residues   measured  in chamise were  221 mg/kg and in  grass and forbs
269 mg/kg within 2 h of application.  After 30 days, these  levels  had
dropped  to 60 mg/kg for chamise and 21 mg/kg for grass and forbs, and,
after  360  days, 0.1  mg/kg was present  in chamise.  Siltanen  et al.
(1981)  monitored residues of 2,4-D in the fruit of bilberries  1  year
after the application of 0.25, 0.75, or 2.25 kg/ha acid equivalent.  No
residues were detected, the limit of detection being 0.05 mg/kg.

    Raatikainen  et  al.  (1979),  in  a  controlled  field experiment,
sprayed  cowberry and  bilberry with  an ester  formulation  of  2,4-D.
Three  application  rates  were used,  0.25,  0.75,  and 2.25  kg  acid
equivalent/ha,  and  residues  of  2,4-D  were  measured  approximately
1 month after application.  Thirty-four days after the  application  of

0.25 kg/ha, residues in cowberry were 0.3 mg/kg.  Cowberries exposed to
0.75 or 2.25 kg/ha were analysed after 35 days and  contained  residues
of  1.0 and  3.7 mg/kg,  respectively.  Bilberries  treated with  0.25,
0.75,  or 2.25 kg/ha  were analysed 29  days later; residues  were 0.1,
1.3, and 4.8 mg/kg, respectively.

4.3  Elimination

    James (1979) studied tissue distribution of 14C-labelled   2,4-D in
the  spiny lobster  (Panulirus argus).  Labelled  herbicide was injected
into  the pericardial  sinus and  animals were  sacrificed  at  regular
intervals.  2,4-D was taken up from the haemolymph, by the green gland,
and excreted unchanged, with an overall half-time of about 8 h.  Tuey &
James  (1980), in a  similar study, found  that the clearance  of 2,4-D
from haemolymph, via the green gland, was three to five  times  greater
than the rate of metabolism in the hepatopancreas.

    Pritchard  & James  (1979) studied  the renal  handling  of  intra-
venously  injected  2,4-D  by the  winter flounder   (Pseudopleuronectes
 americanus).    2,4-D, at a  concentration of 1 µmol/litre   of plasma,
was   actively  secreted  into  the glomerular filtrate  of the  kidney
with   clearances of nearly 500  times the glomerular filtration  rate.
At  higher plasma concentrations  of between 10  and 60 µmol/litre,   a
transport  maximum  of  0.85 µmol/g  of  kidney  per  h  was  observed.
Koschier   &   Pritchard   (1980)  reported  a  similar   study   using
an   elasmobranch   fish     Squalus   acanthias.    They   administered
2.5 µmol   14C-2,4-D/kg    to  the fish  intramuscularly  and monitored
blood and urine 14C   levels.  Clearance of total 2,4-D was  more  than
25 times greater than the glomerular filtration rate,  indicating  that
2,4-D  was being actively secreted by the kidney.  2,4-D was eliminated
in the urine as a taurine conjugate, this representing about 95% of the
excretory   products.   The  plasma contained,  primarily, unconjugated
2,4-D  (>90%).  It seemed,  therefore, that 2,4-D  was conjugated  with
taurine  before being excreted in the urine.  Guarino et al. (1977), in
a  similar  study on  the dogfish  Squalus,  also  found that 2,4-D  was
extensively   conjugated   to   taurine  (>90%)   and   was  eliminated
predominantly via the urine; 70% of the administered dose  appeared  in
the  urine within 4  to 6 days.   The highest  tissue  concentration of
2,4-D  (14.5  mg/kg)  was found  in  the  kidney  after  4  h.   Plasma
elimination  was  rapid, with  a half-time of  44 min; similarly  rapid
clearance was seen from the kidney.  Half-time estimates for muscle and
liver were 2 to 3 days and 5 days, respectively.

5.  TOXICITY TO MICROORGANISMS

 Appraisal

     In  general  2,4-D  is  relatively  non-toxic  to  water  and  soil
 microorganisms at recommended field application rates.

     No  effect of 2,4-D was recorded on 17 genera of freshwater and two
 genera of marine algae at concentrations up to 222 mg/litre.

     No effect of 2,4-D was observed on respiration of either sandy loam
 or clay loam soils at concentrations up to 200 mg/kg.

     N-fixation by aquatic algae is affected at high  concentrations  of
 2,4-D  acid (400 mg/litre).   An effect of  2,4-D esters on  N-fixation
 occurs  from a concentration of 36 mg/litre upwards.  N-fixing algae in
 topsoils  appear to be more  vulnerable to 2,4-D acid  than other algal
 species.   The Cyanobacteria (blue-green  algae) are important  as  the
 major N2  source in tropical ponds and soils.

     In  the range of 25.2 to 50.4 mg/litre, 2,4-D was inhibitory to all
 types of soil fungi.

     Cell division was reduced in a green alga by 2,4-D at  20  mg/litre
 and  stopped  at  50 mg/litre.   No  effect  was observed  on a natural
 phytoplankton   community  after  exposure  to  2,4-D  at  1  mg/litre.
 However,  exposure to  esters of  2,4-D reduced  productivity in  these
 organisms.

5.1.  Aquatic Microorganisms

    Hawxby   et   al.   (1977)   exposed   cultures   of   three  algae
 (Chlorella    pyrenoidosa,   Chlorococcum  sp., and Lyngbya  sp.,)  and
one   cyanobacterium   (blue-green   alga)  (Anabaena  variabilis)    to
concentrations  of  2,4-D  in  the  medium  of  up to  10 µmol   /litre
(=  2.21  mg/litre).  There  was no effect  on growth, respiration,  or
photosynthetic rate.

    Gangawane  et al. (1980) studied the effects of 2,4-D on growth and
heterocyst  formation in the nitrogen-fixing cyanobacterium (blue-green
alga)  Nostoc.    The organism was cultured  for 30 days in  0, 10, 100,
1000, or 1500 mg 2,4-D/litre.  Growth was measured by  optical  density
and  cells forming heterocysts were  counted.  Growth was inhibited  at
both  10  and  100  mg  2,4-D/litre  and  was  eliminated   at   higher
concentrations.  There was also reduced heterocyst formation.

    Lembi  &  Coleridge (1975) demonstrated a marked effect  of  2,4-D,
at   concentrations   of  110   or  220 mg/litre,   on cultures of  the
green  algae  Scenedesmus, Ankistrodesmus,  and  Pediastrum.    After 14
days  of culture, the three  species under control conditions  produced
456 x 102,   634 x 104,   and 227 cells or colonies per ml  of  medium,
respectively.   Corresponding  figures  after exposure  to 110 mg/litre
were  54  x  102,   41  x  104,    and 74  cells  or  colonies per  ml,
respectively.  For  both  Scenedesmus  and  Ankistrodesmus,  these values
were less than the pre-treatment cell concentrations.

    Butler  et al.  (1975b) exposed  unialgal cultures  of green  algae
isolated  from Warrior  River water  to 2,4-D  butoxyethanol  ester  at
0.001,  0.01, 0.1, 1.0, or 4.0 mg/litre.  Thirty separate isolates were
used.   Concentrations less than  or equal to  1 mg/litre of  the 2,4-D
ester did not change the growth pattern of the isolates.  However, with
a  concentration of 4 mg/litre, there was some inhibition of growth, as
indicated  by a 10%  increase in the  number of incubates  which showed
poor  growth, or no growth,  when compared to controls.   Some isolates
were  unaffected  even at  this concentration and  it can therefore  be
assumed  that  2,4-D  butoxyethanol  ester  might  change  the  species
composition of green algae populations.

    Bednarz   (1981)  used  12  pure   cultures  of  green  algae   and
cyanobacteria  (blue-green  algae)  separately and  in  combination  to
investigate  the  effects  of 2,4-D  acid.   Cultures  were exposed  to
concentrations  of  2,4-D  ranging from  0.001  to  10  mg/litre.   Low
concentrations of 2,4-D stimulated the growth of most species of algae,
whereas high concentrations inhibited growth.  Chlorococcal green algae
were  more sensitive  to 2,4-D  than were  filamentous green  algae  or
cyanobacteria.    In   further   experiments,  the   authors   cultured
combinations  of sensitive and  tolerant species in  the same range  of
2,4-D  concentrations.   Tolerant  species used  in  combinations  were
 Chlorella   pyrenoidosa,  Dictyosphaerium pulchellum,  and   Scenedesmus
 quadricaudata.    The first two of  these tolerant species reduced  the
toxicity  of  2,4-D  to  sensitive  species  in  mixed  culture.   This
protective effect was not seen with  Scenedesmus.

    Singh    (1974)    cultured    a   filamentous,    nitrogen-fixing,
cyanobacterium  Cylindrospermum sp.  in concentrations of 2,4-D  acid of
0,  100,  300,  400, 500, 600, 800, 1000, or 1200 mg/litre and examined
growth  and heterocyst formation  after 8 days.   Both parameters  were
affected at concentrations higher than 300 mg/litre and  cultures  were
killed  at a concentration  of 1000 mg/litre.   Kapoor & Sharma  (1980)
exposed  cultures  of  the nitrogen-fixing,  filamentous cyanobacterium
 Anabaena doliolum  to 2,4-D ethyl ester (as `Weedone  48'  concentrate)
at concentrations of 36, 108, 180, 252, or 324 mg/litre.  There  was  a
dose-related  decrease in cell nitrogen  over the whole range  of 2,4-D
ester exposures.  Cell growth was stimulated by lower concentrations of
2,4-D  and only inhibited  by the highest  dose.  Tiwari et  al. (1984)
exposed    cultures   of   a   similar   nitrogen-fixing,   filamentous
cyanobacterium  (Anabaena cylindrica) to 2,4-D acid at concentrations of
0,  100,  500,  700,  1000,  or  1500  mg/litre, and  examined  growth,
heterocyst formation, and nitrogen fixation.  For all these parameters,
there  was  a  stimulatory effect  of  2,4-D  at  100  mg/litre  and  a
progressive  inhibition with higher concentrations.   These and similar
algae  are considered  to be  a major  source of  nitrogen in  tropical
ponds  and soils.   Das &  Singh (1977)  cultured  the  nitrogen-fixing
cyanobacterium  Anaebaenopsis raciborskii  in  concentrations  of  2,4-D
acid  (sodium salt) of 10,  100, 400, 600, 800,  and 1000 mg/litre  and
measured nitrogen fixation.  Control cultures and those exposed  at  10
and   100   mg   2,4-D/litre   showed   no   significant   differences.
Nitrogen-fixation  was inhibited at 400 mg/litre or more and eliminated
at 600 mg/litre.

    Butler  (1963)  reported  no  effect  on  a  natural  phytoplankton
community  of exposure to a  1 mg/litre concentration of  2,4-D (as the
acid  or dimethylamine  salt), or  of the  dimethylamine salt  on  pure
cultures  of  Dunaliella euchlora  or  Platymonas  over 4 h.   In a later
study (Butler, 1965), natural phytoplankton communities were exposed to
esters  of 2,4-D.  Butoxyethanol  ester, propylene glycol  butyl  ether
ester,  and  ethylhexyl ester  reduced  productivity  (as  measured  by
carbon fixation) by 16%, 44%, and 49%, respectively, at a concentration
of 1 mg/litre.

    Sarma  & Tripathi (1980) monitored cell division in the filamentous
green  alga  Oedogonium acmandrium  exposed to 2,4-D  acid at 1, 5,  10,
20,  or  50  mg/litre of culture medium.  At  up  to 10 mg/litre, 2,4-D
was  found  to  stimulate cell division; a 168 h exposure to 5 mg/litre
increased  the  incidence  of dividing  cells  by  15%  over  controls.
However,   cell  division  was  reduced  at 20 mg/litre and  stopped at
50  mg/litre.   Abnormalities  in  chromosomes  during  cell   division
increased with increasing 2,4-D exposure.

    Chai   &   Chung   (1975)   examined   the   effects   on   growth,
photosynthesis,  respiration,  and  chemical  composition  of  exposing
cultures of the green alga  Chlorella ellipsoidea to 2,4-D acid at 22 or
88 mg/litre.  At 22 mg/litre, 2,4-D increased  growth,  photosynthesis,
and  the  cell  content of  protein  and  nucleic acids.   Carbohydrate
content  was unchanged.  However, at 88 mg/litre, growth was inhibited,
photosynthesis  was no different from controls, and the cell content of
carbohydrate, protein, and nucleic acids was decreased.

    Elder  et al. (1970) examined the effect of 2,4-D acid on 17 genera
of   freshwater  and  two genera of marine algae exposed at 22, 111, or
222  mg/litre.   There  was  no  effect on  the  growth of  any  of the
cultures, even at the highest dose of 2,4-D.

    Cultures of the flagellate  Euglena gracilis  were exposed for 24  h
to concentrations of 1, 5, 10, 50, or 100 mg/litre or for 7 days to 10,
50,  or  100  mg/litre of 2,4-D acid by Poorman (1973).  Cultures in 50
and 100 mg 2,4-D/litre yielded 84% and 74%, respectively,  relative  to
controls,  over  24 h.   Lower concentrations of  2,4-D had a  slightly
stimulatory effect.  After 7 days, there was significant stimulation of
yield with 10 mg/litre; the culture yielded 161% compared to a control.
There was slight stimulation of growth by 50 mg/litre and  a  reduction
to 78% of control levels with 100 mg/litre.

    George    et    al.    (1982)   exposed   the  rotifer    Brachionus
 calyciflorus  to 2,4-D at 5 mg/litre.  Median lethal time  (LT50)   was
24 h and LT100 was 31 h.

5.2.  Soil Microorganisms

    Pachpande  &  David (1980)  isolated  the soil  alga    Chlorococcum
 infusionum  from paddy fields and cultured the organism in the presence
of  2,4-D acid at  concentrations of 0,  1, 2, 3,  4, and  5  mg/litre.
Growth was estimated as dry weight of algal cells filtered out  of  the
medium.   All  concentrations  of   2,4-D  were  inhibitory  to growth.
At  the highest 2,4-D concentration  of 5 mg/litre,  the  culture yield
was  reduced  from  a control level of 720 mg dry wt/litre of medium to
520 mg/litre.

    Cullimore  & McCann  (1977) applied  2,4-D acid  to isolated  cores
taken  from a prairie, loam soil to give  approximate concentrations of
1  or  100  mg/kg in the top 2 cm of soil.  Soil algal populations were
estimated  from subsamples of cores taken before treatment and 1, 5, or
20  days after treatment  with herbicide.  Thirty-one  genera of  algae
were  identified, of which five  were very sensitive to  2,4-D and were
rarely  found after treatment.  These were  Chlamydomonas, Chlorococcum,
 Hormidium,  Palmella,  and  Ulothrix.   The  most resistant  genera were
 Chlorella, Lyngbya, Nostoc, and  Hantzschia;  the `percent  sensitivity'
of  these genera (%  of the total  number of treatments  in  which  the
genus  was  absent)  was 28%,  6%,  22%,  and 44%,  respectively.   The
reduction in cell numbers of algae in the top layer of the  soil  after
herbicide treatment was soon offset by an increase in the population of
 Chlorella,  Stichococcus,  Oscillatoria,   and  Spongiochloris,  all  of
which  recovered very rapidly from  the herbicide effects.  There  was,
however, an overall reduction in cell numbers of nitrogen-fixing algae.

    Mukhopadhyay   (1980)   measured   the   bacterial,   fungal,   and
actinomycete populations of soils supporting rice or maize plants which
had been treated with various herbicides for weed control.   There  was
no  effect of 2,4-D,  applied at the  recommended rate, either  on soil
microorganism  numbers or on  the evolution of  carbon dioxide by  soil
cultures.

    Huber  et  al.  (1980) examined the effect of 2,4-D at 0.3, 0.2, or
0.1  mmol/litre (=  66, 44,  and 22  mg/litre, respectively)  on  seven
cultures  of soil microorganisms.  There was no effect on the growth of
five  of the cultures; these were  Nocardia sp., Pseudomonas fluorescens
in   both  aerobic  and  anaerobic  culture,  Bacillus  subtilis,    and
 Ustilago   maydis.    There  was a  small  reduction  in growth  at the
highest  2,4-D dose in cultures of  Rhizopus japonicus  and   Aspergillus
 niger.   2,4-D  had no effect on mycelium growth of three out  of  four
plant  pathogenic  fungi  in  culture;  Phytophthora  cryptogea   showed
reduced  mycelial  growth  at  0.1,  0.2,  and  0.3  mmol  2,4-D/litre,
but Fusarium   oxysporum,  Alternia  radicina,  and  Rhizoctonia  solani
were unaffected.

    Moubasher et al. (1981) added 2,4-D at three doses (1.9,  7.6,  and
15.2  mg/kg) either  to soil  or to  agar medium  inoculated with  soil
fungi,  and the effects on fungal populations were monitored.  In soil,
at all three doses, 2,4-D stimulated the fungi.  When  incorporated  in
the  agar medium, 2,4-D was stimulatory to overall fungal growth and to
four individual species of fungus at the lowest dose of  6.3  mg/litre,
but   inhibitory  to  two  other species.  At  higher doses of  25.2 or
50.4 mg/litre, the herbicide was inhibitory to all fungi.

    2,4-D  had a significant inhibitory effect on culture yields of the
bacterium  Escherichia  coli only  at 10-3mol/litre    (= 220 mg/litre).
There  was no effect at 10-4mol/litre   (= 22 mg/litre) (Toure & Stenz,
1977).

    Prescot & Olson (1972) added 2,4-D, at doses of 0, 0.1, 1.0, 10, or
100 mg/litre, to cultures of the soil  amoeba  Acanthamoeba  castellanii
and  monitored growth and reproduction.  There was a stimulatory effect
of  2,4-D at all dose levels; this effect was most marked at the lowest
dose  and declined  with increasing  exposure to  2,4-D.   The  authors

suggest  that the amoeba  may degrade the  2,4-D and utilize  it  as  a
carbon source.  However, Pons & Pussard (1980) found no effect of 2,4-D
(at 28, 54, or 84 mg/litre) on the reproduction of 23 different strains
of free-living soil amoebae.

    2,4-D, at 10-3mol/litre   in cultures of the ascomycete   Neurospora
 crassa,    stimulated   DNA  synthesis  but  had  no  effect  at  lower
concentrations  of 10-4   to 10-6mol/litre.    These concentrations had
no  significant  effect  on either  RNA  or  protein (Schroder  et al.,
1970).

    Naguib et al. (1980) measured growth, respiration,  and  absorption
and  utilization  of sugar  and nitrogen in  pre-formed fungal mats  of
 Aspergillus  terreus  over  72 h   in the presence  of 200 mg/litre  of
2,4-D.   The herbicide inhibited sugar inversion and consequently sugar
absorption. It also reduced the incorporation of nitrogen into protein.
Respiration was depressed.  Growth of the fungus was suppressed and, on
a  dry weight  basis, culture  mass was  reduced to  below the  initial
level.

    Trevors   &  Starodub  (1983)  added 2,4-D to  sandy loam and  clay
loam   soils   and  measured  both  respiration and  electron transport
system  (ETS)  activity.  ETS  was assessed by measuring  the  capacity
of the  soil  to  reduce  2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl
tetrazolium   chloride  (INT)  to  iodonitrotetrazolium  formazan  (INT
formazan).  The effects of 2,4-D were tested at concentrations  of  the
herbicide in soil of 0, 10, 25, 50, 75, 100, or 200 mg/kg.   There  was
no effect on soil respiration, monitored either as  oxygen  consumption
or carbon dioxide evolution, at any of the concentrations of  2,4-D  in
either   soil.  There was similarly no effect on ETS in the sandy loam.
However,  in the clay loam,  there was a progressive  inhibition of ETS
over the whole range of concentrations of the herbicide.   The  control
soil  had an ETS activity  of 37.3 µg  INT formazan  production/g soil,
whereas  the  ETS  activity of  soil  treated  with 10  mg 2,4-D/kg was
25.1 µg   INT   formazan/g,   significantly  lower  than  that  of  the
control.    The   activity   was  reduced   further   with   increasing
concentrations of 2,4-D, until an activity of 16.3 µg   INT  formazan/g
was found at 200 mg 2,4-D/kg.

    Deshmukh  &  Shrikhande (1975)  added  2,4-D, at  recommended field
rates,   and   at  five   times  the  recommended  field rates, to  two
types  of  soil  from India.   Both  doses  of 2,4-D  inhibited numbers
of  Azobacter in both soil types, and the high, but not the low, dose of
2,4-D  reduced  nitrogen  fixation in  both  soils.   The same  authors
(Deshmukh  &  Shrikhande, 1974)  monitored  the populations  of various
microorganisms  under  the  same dosing  conditions.   2,4-D stimulated
the numbers of actinomycetes throughout the 6-week incubation period at
both dose levels.  Fungal populations were reduced in the first week of
incubation  at  both  dose levels in sandy loam, but only at the higher
dose  level  in  clay  loam.   This  reduction  in  fungal  populations
persisted  until  the  second week with the high dose in the sandy soil
and throughout the incubation period with the high dose in  clay  soil.
There  was a temporary  (1 week) reduction  in total bacterial  numbers
with both 2,4-D dose levels in the sandy soil and with the higher level
in   clay  soil.   Schroder  &  Pilz  (1983)  reported  that  2,4-D  at
approximately 10-4mol/litre   (= 22 mg/kg) had no long-term  effect  on
soil nitrification.

    Welp  &  Brummer  (1985) measured  the  influence  of 2,4-D  on the
reducing  capacity of soil microorganisms, reduction being monitored as
the  capacity to reduce  Fe(III) oxides to  soluble Fe(II) ions.   They
determined no-observed-effect levels (NOEL) of 115 and 95  mg  2,4-D/kg
for   two  different  soil  types  and  corresponding  EC50 values   on
reduction capacity of 200 and 530 mg 2,4-D/kg soil.

    Ruggiero  & Radogna (1985)  extracted and partially  purified  soil
diphenolase (laccase) from forest soil.  This enzyme, which exists free
in  the  soil,  plays an  important  role  in the  metabolism  of humic
materials  in  soil.   Oxygen  consumption  was  monitored  during  the
enzymatic reaction,  using  either catechol  or  p -phenylenediamine  as
substrate,  and the effect  of 2,4-D was  investigated.  The  herbicide
inhibited   diphenolase   activity,   and Lineweaver-Burk  plots of the
data   suggested  that  2,4-D  acts  as  a  non-competitive  inhibitor.
Apparent  K values of 28.7 and 6.0 mol/litre were obtained for catechol
and  p -phenylenediamine, respectively.

6.  TOXICITY TO AQUATIC ORGANISMS

6.1.  Toxicity to Aquatic Invertebrates

 Appraisal

     The  short-term toxicity data  on the effects  of 2,4-D free  acid,
 its  salts, and esters  on aquatic invertebrates  is extensive.   Ester
 formulations  are more toxic than the free acids or salts.  Sensitivity
 variations  exist among species  in response to  the same  formulation.
 Organisms  become more sensitive  to 2,4-D when  the water  temperature
 increases.   Reproductive  impairment occurred  at concentrations below
 0.1 of the short-term toxic levels determined for these formulations.

6.1.1.  Short-term toxicity

    The  short-term  toxicity  of  2,4-D  to  aquatic  invertebrates is
summarized in tables 3 - 5.

    Unfortunately, there are few studies where both the free  acid  (or
its salts) and ester preparations have been tested on the same organism
under the same conditions.  The only organisms for which  this  applies
are  the oyster (Butler, 1963;  Butler, 1965), the stonefly  (Sanders &
Cope, 1968), and daphnids and shrimp (Sanders, 1970a).   These  studies
all  show that the free  acid and its salts  are less toxic than  ester
formulations; for example the free acid is at least 20 times less toxic
to  the  water flea  Daphnia magna  than the least  toxic of the  esters
tested  (Sanders, 1970a).  Comparing  studies carried out  by different
authors  and in different systems also suggests a much greater toxicity
of the ester preparations.

    Liu  & Lee (1975) found  that 2,4-D could adversely  affect the bay
mussel  (Mytilus edulis) at all stages of its life cycle. The attachment
of young mussels to test chamber walls was reduced (data in  Table  3).
The  authors evaluated, in two  duplicate experiments, the  effects  of
2,4-D  acid, at concentrations  in sea water  of 22.8, 45.7,  91.4, and
182.8  mg/litre,  on  the growth  of  larval  mussels.  After  10  days
exposure,  there was a  significant reduction in  the growth of  larvae
exposed  to  91.4  mg  2,4-D/litre;  larvae  were  11.6%  smaller  than
controls.  This reduction was found in only one experimental replicate.
In both  experiments, there was reduced growth after 10  days  exposure
to 182.8 mg/litre;  larvae were 31.9% and 34.9% smaller  than  controls
in   the  two  experiments.  Exposure   for  20  days at  91.4 mg/litre
led   to  reduced  growth in  both experiments.  All larvae  exposed to
182.8  mg/litre  died  within 12  days,  but  only in  one experimental
replicate.   Extension of the growth study in the second experiment led
to all larvae dying within 22 days of exposure to 182.8  mg/litre  and,
therefore,  failing  to  undergo metamorphosis.   The  metamorphosis of
larvae  exposed from age  30 to 70  days was not  affected by 2,4-D  at
concentrations up to 176 mg/litre.

    Presing (1981) monitored reproduction over four broods in the water
flea Daphnia  magna exposed  to  0,  5,  10,  25,  or  50  mg/litre  of
`Dikonirt'  (sodium salt  of 2,4-D).   For the  first brood,  the  only
significant  effect was at  50 mg/litre, whereas  the fourth brood  was
delayed  even  at  5 or  10  mg/litre.   Significant reductions  in the
average   number  of  young  produced  for each female were  found with
the  two highest concentrations.   Young  kept  until   maturity   from
each  of   the  tests were  themselves  exposed  to 2,4-D  in  a repeat
experiment.   Again there was a significant effect on young produced at
25 and 50 mg/litre.


Table 3.  Toxicity of 2,4-D to estuarine or marine invertebrates
---------------------------------------------------------------------------------------------------------
Organism                   Flow/  Temp  Salinity  pH   Formulationc    Parameter      Water      Reference
                           stata  (°C)  (o/oo)                                     concentration
                                                                                    (mg/litre)
---------------------------------------------------------------------------------------------------------
Bay mussel                        17.2-  22.9-    6.4-  free acid      96-h LC50      259        Liu & 
 (Mytilus edulis)                  18.6   24.5     7.8                              (232-289)     Lee (1975)
                                  17.2-  22.9-    6.4-  free acid      96-h EC50      262        Liu & 
                                  18.6   24.5     7.8                  attachment                Lee (1975)
    (trocophore larva)             17.2-  22.9-    6.4-  free acid      48-h EC50      211.7      Liu & 
                                  18.6   24.5     7.8                  normal                    Lee (1975)
                                                                       development

Eastern oyster             flow   18     29             butoxyethanol  96-h EC50        3.75     Butler 
 (Crassostrea virginica)                                                shell growth              (1963)
                           flow   29     25             isooctyl       96-h EC50        1.0      Mayer 
                                                                       shell growth              (1987)
                           flow   28     25             PGBEE          96-h EC50        0.055    Mayer 
                                                                       shell growth              (1987)

Copepod                           21      7       7.8   butoxyethanol  96-h LC50        3.1      Linden 
 (Nitocra spinipes)                                                                  (2.4-4.1)    et al. 
                                                                                                 (1979)
Brown shrimp (adult)       flow   30                    PGBEE          24-h EC50        0.55     Butler 
 (Penaeus aztecus)                                                      loss of                   (1963)
                                                                       equilibrium
    (adult)                 flow   30                    PGBEE          48-h EC50        0.55     Butler 
                                                                       loss of                   (1963)
                                                                       equilibrium
   (juv.)b                 stat   26     30             butoxyethanol  48-h LC50        5.6      Mayer 
                                                                                                 (1987)
    (adult)                 flow   29     26             isooctyl       48-h LC50        0.48     Mayer 
                                                                                                 (1987)
Dungeness crab (1st zoel)  stat   13     25             acid (tech)    96-h LC50     > 10        Caldwell 
 (Cancer magister)                                                                                (1977)
   (1st instar juv.)b      stat   13     25             acid (tech)    96-h LC50     > 100       Caldwell 
                                                                                                 (1977)
Blue crab     (juv.)b      stat   24     29             PGBEE          48-h LC50        2.8      Mayer 
 (Callinectes sapidus)                                                                            (1987)
---------------------------------------------------------------------------------------------------------
a  Stat = static conditions (water unchanged for duration of test); flow = flow-through conditions 
   (2,4-D concentration
   in water continuously maintained.
b  juv. = juvenile.
c  PGBEE = propylene glycol butyl ethyl ester.

Table 4.  Toxicity of 2,4-D to freshwater invertebrates
---------------------------------------------------------------------------------------------------------
Organism             Flow/  Temp  Alkali- Hard-  pH    Formulationd   Parameter    Water       Reference
                     stata  (°C)  nityb   nessb                                 concentration
                                                                                  (mg/litre)
---------------------------------------------------------------------------------------------------------
Oligochaete worm     flow   20    30      30     7.8   free acid      48-h LC50  122.2         Bailey &
 (Lumbriculus         flow   20    30      30     7.8   free acid      96-h LC50  122.2         Liu (1980)
  variegatus)

Water flea           stat   21    260     272    7.4   PGBEE          48-h LC50  0.1           Sanders (1970a)
 (Daphnia magna)      stat   21    260     272    7.4   dimethylamine  48-h LC50  4.0           Sanders (1970a)
                     stat   17            39     7.2   dimethylamine  48-h LC50  > 100.0       Mayer &
                                                                                               Ellersieck(1986)
                     stat   21    260     272    7.4   butoxyethanol  48-h LC50  5.6           Sanders (1970a)
                     stat   21    260     272    7.4   free acid      48-h LC50  > 100.0       Sanders (1970a)
                            20                   8.4-  free acid      96-h LC50  417.8         Presing (1981)
                                                 8.6
                            20                   8.4-  sodium salt    96-h LC50  932.1         Presing (1981)
                                                 8.6

Water flea                  15.6          44     7.4   PGBEE          48-h LC50  4.9           Sanders &
 (Simocephalus                                                                    (4.0-6.7)     Cope (1966)
 serrulatus)

Water flea                  15.6                       PGBEE          48-h LC50  3.2           Sanders &
(Daphnia pulex)                                                                  (2.4-4.3)     Cope (1966)

Copepod (nauplius larva)
(Cyclops vernalis)   stat   20    31.6    70     6.7   free acid      96-h LC50  8.72          Robertson (1975)
                                                                                 (5.34-11.57)
                     stat   20    31.6    70     6.7   alkanolamine   96-h LC50  54.8          Robertson (1975)
                                                                                 (46.45-64.6)

Scud                 stat   21.1  30             7.1   butoxyethanol  24-h LC50  1.4 (1.1-1.8) Sanders (1969)
 (Gammarus            stat   21.1  30             7.1   butoxyethanol  48-h LC50  0.76 (0.51
  lacustris)                                                                      -1.1)         Sanders (1969)
                     stat   21.1  30             7.1   butoxyethanol  96-h LC50  0.44 (0.31
                                                                                 -0.62)        Sanders (1969)
                     stat   21.1  30             7.1   PGBEE          24-h LC50  2.1 (1.7-2.5) Sanders (1969)
                     stat   21.1  30             7.1   PGBEE          48-h LC50  1.8 (1.4-2.3) Sanders (1969)
                     stat   21.1  30             7.1   PGBEE          96-h LC50  1.6 (1.2-2.1) Sanders (1969)
                     stat   21.1  30             7.1   isooctyl       24-h LC50  6.8 (4.8-9.7) Sanders (1969)
                     stat   21.1  30             7.1   isooctyl       48-h LC50  4.6 (2.9-7.3) Sanders (1969)
                     stat   21.1  30             7.1   isooctyl       96-h LC50  2.4 (1.9-4.8) Sanders (1969)
                     stat   15.5  260     272    7.4   PGBEE          24-h LC50  4.1 (2.8-5.8) Sanders (1970a)
                     stat   15.5  260     272    7.4   PGBEE          48-h LC50  2.6 (1.7-3.9) Sanders (1970a)
---------------------------------------------------------------------------------------------------------

    Table 4.  (contd.)
---------------------------------------------------------------------------------------------------------
Organism             Flow/  Temp  Alkali- Hard-  pH    Formulationd   Parameter     Water      Reference
                     stata  (°C)  nityb   nessb                                  concentration
                                                                                  (mg/litre)
---------------------------------------------------------------------------------------------------------
Scud                 stat   15.5  260     272    7.4   PGBEE          96-h LC50  2.5 (1.7-3.7) Sanders (1970a)
 (Gammarus            stat   15.5  260     272    7.4   butoxyethanol  24-h LC50  6.5 (1.0-8.6) Sanders (1970a)
  lacustris) (contd.) stat   15.5  260     272    7.4   butoxyethanol  48-h LC50  5.9 (3.1-11)  Sanders (1970a)
                     stat   15.5  260     272    7.4   butoxyethanol  96-h LC50  5.9 (3.1-11)  Sanders (1970a)

Scud                 stat   15            272    7.4   dimethylamine  24-h LC50  > 100         Mayer &
 (Gammarus fasciatus) stat   15            272    7.4   dimethylamine  96-h LC50  > 100         Ellersieck (1986)

Glass shrimp         stat   21    260     272    7.4   PGBEE          48-h LC50  2.7           Sanders (1970a)
 (Palaemonetes        stat   21    260     272    7.4   dimethylamine  48-h LC50  > 100         Sanders (1970a)
  kadiakensis         stat   21    260     272    7.4   butoxyethanol  48-h LC50  1.4           Sanders (1970a)

Seed shrimp          stat   21    260     272    7.4   PGBEE          48-h LC50  0.32          Sanders (1970a)
 (Cypridopsis vidua)  stat   21    260     272    7.4   dimethylamine  48-h LC50  8.0           Sanders (1970a)
                     stat   21    260     272    7.4   butoxyethanol  48-h LC50  1.8           Sanders (1970a)

Freshwater prawn     stat   27            113.9  7.5   sodium salt    24-h LC50  2342          Shukla &
 (Macrobranchium      stat   27            113.9  7.5   sodium salt    48-h LC50  2309          Omkar (1983)
  lamarrei)           stat   27            113.9  7.5   sodium salt    72-h LC50  2267          Shukla &
                     stat   27            113.9  7.5   sodium salt    96-h LC50  2224          Omkar (1983)

Freshwater prawn     stat   28            112.7  7.5   sodium salt    24-h LC50  2644          Omkar &
 (Macrobranchium      stat   28            112.7  7.5   sodium salt    48-h LC50  2536          Shukla (1984)
  naso)               stat   28            112.7  7.5   sodium salt    72-h LC50  2435          Omkar &
                     stat   28            112.7  7.5   sodium salt    96-h LC50  2397          Shukla (1984)

Freshwater prawn     stat   28            112.7  7.5   sodium salt    24-h LC50  2474          Omkar &
 (Macrobranchium      stat   28            112.7  7.5   sodium salt    48-h LC50  2381          Shukla (1984)
  dayanum)            stat   28            112.7  7.5   sodium salt    72-h LC50  2333          Omkar &
                     stat   28            112.7  7.5   sodium salt    96-h LC50  2275          Shukla (1984)

Crayfish             stat   15.5  260     272    7.4   PGBEE          48-h LC50  > 100         Sanders (1970a)
 (Orconectes nais)    stat   15.5  260     272    7.4   dimethylamine  48-h LC50  > 100         Sanders (1970a)
                     stat   15.5  260     272    7.4   butoxyethanol  48-h LC50  > 100         Sanders (1970a)

Red swamp            stat   20            100    8.4   alkanolamine   96-h LC50  1389          Cheah et al.
crayfish (imm.)c                                                                 (1174-1681)   (1980)
 (Procambarus clarki)

---------------------------------------------------------------------------------------------------------

    Table 4.  (contd.)
---------------------------------------------------------------------------------------------------------
Organism             Flow/  Temp  Alkali- Hard-  pH    Formulationd   Parameter     Water      Reference
                     stata  (°C)  nityb   nessb                                  concentration
                                                                                   (mg/litre)
---------------------------------------------------------------------------------------------------------
Sowbug               stat   15.5  260     272    7.4   PGBEE          48-h LC50  2.2           Sanders (1970a)
 (Asellus             stat   15.5  260     272    7.4   dimethylamine  48-h LC50  > 100         Sanders (1970a)
  brevicaudus)        stat   15.5  260     272    7.4   butoxyethanol  48-h LC50  3.2           Sanders (1970a)

Stone fly  (naiad)          15.5  35             7.1   butoxyethanol  24-h LC50  8.5 (5.7-13)  Sanders &
 (Pteronarcys                15.5  35             7.1   butoxyethanol  48-h LC50  1.8 (1.5-2.7) Cope (1968)
  californica)               15.5  35             7.1   butoxyethanol  96-h LC50  1.6 (1.3-1.9) Sanders &
                            15.5  35             7.1   acid (tech)    24-h LC50  56 (50-63)    Cope (1968)
                            15.5  35             7.1   acid (tech)    48-h LC50  44 (32-59)    Sanders &
                            15.5  35             7.1   acid (tech)    96-h LC50  15 (10-22)    Cope (1968)
                                            
Midge  (larva)              15    78-95   55  7.3-7.8  dimethylamine  24-h LC50  1490          Bunting &
 (Chaoborus                  15    78-95   55  7.3-7.8  dimethylamine  96-h LC50  890 (421-1211)Robertson
 punctipennis)               20    78-95   55  7.3-7.8  dimethylamine  24-h LC50  1124          (1975)
---------------------------------------------------------------------------------------------------------
a  Stat = static conditions (water unchanged for duration of test); flow = flow-through conditions 
   (2,4-D concentration in water continuously maintained.
b  Alkalinity and hardness expressed as mg CaCO3/litre.
c  imm. = immature.
d  PGBEE = propylene glycol butyl ether ester.

    Table 5.  Toxicity of 2,4-D to aquatic invertebrates: no observed effect levels
---------------------------------------------------------------------------------------------------------
                       Flow/ Temp  Sali- Alkali- Hard-                                Water con-  Refer-
Organism               stata (°C)  nity  nityb   nessb  pH  Formulationc  Parameterd  centration  ence
                                  (o/oo)                                              (mg/litre)         
---------------------------------------------------------------------------------------------------------
Eastern oyster         flow   9    19                       free acid     96-h EC0      2.0       Butler 
 (Crassostrea                                                              shell growth            (1963)
  virginica)            flow   30   23                       free acid     96-h EC0      2.0       Butler 
                                                                          shell growth            (1963)
                       flow   25   28                       dimethylamine 96-h EC0      2.0       Butler 
                                                                          shell growth            (1963)

Freshwater oligochaete flow   20         30      30    7.8  free acid     96-h LC0      86.7      Bailey 
 (Lumbriculus                                                                                      & Liu
  variegatus)                                                                                      (1980)

Scud                   stat   21.1       30            7.1  dimethylamine 96-h LC0      100       Sanders 
 (Gammarus lacustris)                                                                              (1969)

Grass shrimp           stat   20   20                       butoxyethanol 24-h LC0      10        Hansen 
 (Palaemonetes pugio)                                                                              et al.
                                                                                                  (1973)
Pink shrimp                                                 butoxyethanol 48-h LC0      1.0       Butler 
 (Penaeus duorarum)                                                                                (1965)
                                                            PGBEE         48-h LC0      1.0       Butler 
                                                                                                  (1965)
---------------------------------------------------------------------------------------------------------
a  Stat = static conditions (water unchanged for duration of test); flow = flow-through conditions 
   (2,4-D concentration in water continuously maintained).
b  Alkalinity and hardness expressed as mg CaCO3/litre.
c  PGBEE = propylene glycol butyl ether ester.
d  LC0 and EC0 represent the highest dose used which cause no death or no effect, respectively; 
   they are not mathematically determined no-effect levels.
    George et al. (1982) measured lethal times (LT) after  exposure  of
the  water flea  Daphnia lumholtzi  to 10  or 20  mg 2,4-D/litre.   They
reported, for 10 mg/litre, an LT50 of  38 h and an LT100 of  71 h.  For
20  mg/litre,  the LT50 was   21 h and  the LT100 was  31  h.  Doses of
2,4-D  ranging from 0.1 to 50 mg/litre did not affect the behaviour of,
or  kill,  the  copepod  Mesocyclops leuckarti  within a 30-day exposure
period and so lethal times could not be calculated.

    Caldwell (1977) and Caldwell et al. (1979) found the zoeal larva to
be the most sensitive life-cycle stage of the Dungeness  crab    (Cancer
 magister)  to the free acid of 2,4-D. Based on the herbicide's toxicity
to  this stage, the authors suggest a maximum acceptable toxicant level
(MATC)  of <1 mg/litre.  At this concentration, there was no mortality,
but there was an effect on moulting.

6.1.2.  Behavioural effects

    Folmar (1978) tested mayfly nymphs  (Ephemerella walkeri)  in a `Y'-
shaped  avoidance maze.  A  2,4-D dimethylamine salt  solution was  run
into  one arm of the  maze and clean water  was run into a  second arm,
both  at 400 ml/min.  Numbers  of nymphs in each  arm of the maze  were
counted after 1 h.  No avoidance of 2,4-D was found  at  concentrations
of 10 mg/litre and there was no mortality.  At 100 mg/litre  there  was
70%  mortality  in  the test  nymphs  but  still no  avoidance  of  the
herbicide.    In   a  similar   experiment   using  the   grass  shrimp
 (Palaemonetes  pugio)  exposed  to  the butoxyethanol  ester  of 2,4-D,
there  was significant avoidance of the herbicide at 1 mg/litre (Hansen
et al., 1973).

6.2.  Toxicity to Fish

 Appraisal

     At  recommended application rates,  the concentration of  2,4-D  in
 water  has  been  estimated to  be  a  maximum of  50  mg/litre.   Most
 applications  would lead to water  concentrations much lower than  this
 (between 0.1 and 1.0 mg/litre).

    LC50  values   for fish vary considerably.  This variation is due to
 differences  in species sensitivity, chemical structure (esters, salts,
 or free acid), and formulation of the herbicide.

     Although   the free acid is  the physiologically toxic entity,  the
 ester  formulations represent a major hazard to fish when used directly
 as aquatic herbicides (because they are more readily taken up by fish).
 Amine  salt formulations used  to control aquatic  weeds do not  affect
 adult fish.

     The  NOEL varies with the species and the  formulation: <1 mg/litre
 (coho salmon) to 50 mg/litre (rainbow trout).

     Fish larvae are the most sensitive life stage but are  unlikely  to
 be affected under normal usage of the herbicide.

     Long-term   adverse   effects  on   fish   are  observed   only  at
 concentrations  higher than those produced after 2,4-D has been applied
 at recommended rates.

     Few  studies are related to the effects of environmental variables,
 such  as  temperature and  water hardness, on  2,4-D toxicity to  fish.
 Higher  temperature  possibly increases  the  toxicity.  This  might be
 considered when assessing the safety of 2,4-D to fish during control of
 aquatic weeds.

     Fish  detect and  avoid 2,4-D  only at  higher concentrations  than
 those obtained under normal conditions of use.

6.2.1.  Effect of formulation on short-term toxicity to fish

    The  toxicity  of  different  formulations  of  2,4-D  to  fish  is
summarized in Table 6.

    The   most  comprehensive  study   on  the  effects   of  different
formulations  of 2,4-D using  the same test  fish, fingerling  bluegill
sunfish  (Lepomis  macrochirus), was performed by Hughes  & Davis (1963)
in static 24-h and 48-h tests.  Ester formulations were invariably more
toxic  than  amine  salt formulations.   Dimethylamine and alkanolamine
preparations ranged in toxicity from 166 to 900 mg/litre (LC50 in  24-h
tests),  depending on the commercial preparation used.  Although esters
were  always more  toxic than  amine salts,  there was  some  variation
between different ester formulations (range: 0.9 to 66.3 mg/litre; 24-h
LC50).     Most of this variation was between different preparations of
the  least  toxic of  the esters, the  isooctyl ester, which  ranged in
toxicity from 8.8 to 66.3 mg/litre.  All other esters  tested  produced
LC50 values   of 8 mg/litre or less, the most toxic being the isopropyl
with a 24-h LC50 of  0.9 mg/litre.  The addition of emulsifiers to acid
preparations  increased 2,4-D toxicity; a  formulation with emulsifiers
gave  an  LC50 of  8  mg/litre over 24  h, making it  comparable to the
esters  in toxicity.   All ester  formulations were  considered by  the
authors  to  present a  major hazard to  fish when used  directly as an
aquatic  herbicide, whereas the amine salt formulations could be safely
used  to control aquatic weeds  without adversely affecting adult  fish
(Hughes & Davis, 1963).

    A study on a range of ester formulations, using salmonids  as  test
fish,  conducted by Finlayson & Verrue (1985), showed that the toxicity
for  salmonids was similar to that for bluegill sunfish.  These authors
argue  that static  tests underestimate  the toxicity  of 2,4-D  esters
because  some of the  ester is hydrolysed  to the less-toxic  free acid
during  the course of even short-term tests.  The presence of test fish
increases  the rate of hydrolysis  of 2,4-D esters.  In  a static test,
with  two different stocking  rates of fish,  the apparent toxicity  of
2,4-D  ester  decreased with  a greater density  of test fish  (rainbow
trout)   because of  this enhanced  hydrolysis.  Results  are given  in
Table  6.  In their flow-through  tests, results were adjusted  to take
account of the hydrolysis of ester to 2,4-D acid during the  course  of
the  experiment.   Two  values are given in Table 6 for each test.  The
first  is  the calculated  effect of the  non-hydrolysed ester and  the
second, entered as `total 2,4-D', is the observed effect of the mixture
of  ester and free acid produced by hydrolysis during the course of the
experiment.  There is as much as a five-fold difference between the two
values.  Alabaster (1969) examined several formulations of 2,4-D in two
species  of fish, and  found that pelleted  herbicide, either as  clay-
based or resin-based pellets, was the least toxic to fish of any of the
formulations tested.


Table 6.  Toxicity of 2,4-D to fish: effects of different formulations
---------------------------------------------------------------------------------------------------------
Organism                 Flow/ Temp Alkali- Hard- pH   Formulationc   Parameter   Water      Reference
                         stata (°C) nityb   nessb                              concentration
                                                                                (mg/litre)
--------------------------------------------------------------------------------------------------------
Bluegill sunfish         stat  25    40      29   6.9  alkanolamine   24-h LC50   450-900  Hughes &
 (Lepomis macrochirus)    stat  25    40      29   6.9  alkanolamine   48-h LC50   435-840  Davis (1963)
                         stat  25    40      29   6.9  dimethylamine  24-h LC50   166-542  Hughes &
                         stat  25    40      29   6.9  dimethylamine  48-h LC50   166-458  Davis (1963)
                         stat  25    40      29   6.9  di-N,N         24-h LC50   1.5      Hughes &
                         stat  25    40      29   6.9  di-N,N         48-h LC50   1.5      Davis
                         stat  25    40      29   6.9  2,4-D acid +   24-h LC50   8.0      (1963)
                                                       emulsifiers
                         stat  25    40      29   6.9  2,4-D acid +   48-h LC50   8.0      Hughes &
                                                       emulsifiers                         Davis (1963)
                         stat  25    40      29   6.9  isooctyl ester 24-h LC50   8.8-66.3 Hughes &
                         stat  25    40      29   6.9  isooctyl ester 48-h LC50   8.8-59.7 Davis (1963)
                         stat  25    40      29   6.9  PGBEE          24-h LC50   2.1      Hughes &
                         stat  25    40      29   6.9  PGBEE          48-h LC50   2.1      Davis (1963)
                         stat  25    40      29   6.9  butoxyethanol  24-h LC50   2.1      Hughes &
                         stat  25    40      29   6.9  butoxyethanol  48-h LC50   2.1      Davis (1963)
                         stat  25    40      29   6.9  butyl ester    24-h LC50   1.3      Hughes &
                         stat  25    40      29   6.9  butyl ester    48-h LC50   1.3      Davis (1963)
                         stat  25    40      29   6.9  mixed butyl +  24-h LC50   1.7      Hughes &
                                                       isopropyl esters                    Davis (1963)
                         stat  25    40      29   6.9  mixed butyl +  48-h LC50   1.7      Hughes &
                                                       isopropyl esters                    Davis (1963)
                         stat  25    40      29   6.9  isopropylester 24-h LC50   0.9      Hughes &
                         stat  25    40      29   6.9  isopropylester 48-h LC50   0.8      Davis (1963)
                         stat  25    40      29   6.9  ethyl ester    24-h LC50   1.4      Hughes &
                         stat  25    40      29   6.9  ethyl ester    48-h LC50   1.4      Davis (1963)

Cutthroat trout                                        butyl ester    96-h LC50   0.78     Woodward (1982)
(juvenile)  (Salmo clarki)                                                       (0.66-0.92)
                                                       PGBEE          96-h LC50   0.77     Woodward (1982)
                                                                                (0.62-0.96)
                                                       isooctyl ester 96-h LC50   > 50    Woodward (1982)

Chinook salmon (fry)     flow  9     18      17   7.1  butoxyethanol  96-h LC50   0.315    Finlayson &
 (Oncorhynchus            flow  9     18      17   7.1  total 2,4-D    96-h LC50   0.373    Verrue (1985)
  tshawytscha)
               (smolts)  flow  15    18      17   7.1  butoxyethanol  96-h LC50   0.375    Finlayson &
                         flow  15    18      17   7.1  total 2,4-D    96-h LC50   1.250    Verrue (1985)
                         flow  15    18      17   7.1  PGBEE          96-h LC50   0.246    Finlayson &
                         flow  15    18      17   7.1  total 2,4-D    96-h LC50   1.117    Verrue (1985)
---------------------------------------------------------------------------------------------------------

Table 6.  (contd.)
---------------------------------------------------------------------------------------------------------
Organism                 Flow/ Temp Alkali- Hard- pH   Formulationc   Parameter   Water      Reference
                         stata (°C) nityb   nessb                              concentration
                                                                                (mg/litre)
---------------------------------------------------------------------------------------------------------
Rainbow trout  (fry)     flow  15    18      17   7.1  butoxyethanol  96-h LC50   0.518    Finlayson &
 (Salmo gairdneri)        flow  15    18      17   7.1  total 2,4-D    96-h LC50   0.642    Verrue (1985)
                         flow  15    18      17   7.1  PGBEE          96-h LC50   0.329    Finlayson &
                         flow  15    18      17   7.1  total 2,4-D    96-h LC50   0.514    Verrue (1985)
               (smolts)  flow  15    18      17   7.1  butoxyethanol  96-h LC50   0.468    Finlayson &
                         flow  15    18      17   7.1  total 2,4-D    96-h LC50   1.338    Verrue (1985)
                         flow  15    18      17   7.1  PGBEE          96-h LC50   0.342    Finlayson &
                         flow  15    18      17   7.1  total 2,4-D    96-h LC50   1.555    Verrue (1985)
    loading factor       stat  14    18      17   7.1  butoxyethanol  96-h LC50   1.206    Finlayson &
    4.2 g fish/litre                                                                       Verrue (1985)
                         stat  14    18      17   7.1  total 2,4-D    96-h LC50   1.422    Finlayson &
    loading factor       stat  15    18      17   7.1  butoxyethanol  96-h LC50   3.689    Verrue (1985)
    8.8 g fish/litre                                                                       Finlayson &
                         stat  15    18      17   7.1  total 2,4-D    96-h LC50   4.487    Verrue (1985)

Harlequin fish           flow  20            250  7.2  clay-based     24-h LC50   7000     Alabaster (1969)
 (Rasbora heteromorpha)                                 pellets
                         flow  20            250  7.2  resin-based    24-h LC50   3950     Alabaster (1969)
                                                       pellets
                         flow  20            250  7.2  resin-based    48-h LC50   3100     Alabaster (1969)
                                                       pellets
                         flow  20            20   7.2  sodium salt    24-h LC50   1160     Alabaster (1969)
                         flow  20            20   7.2  butoxyethyl    24-h LC50   1.0      Alabaster (1969)
                         flow  20            20   7.2  butoxyethyl    48-h LC50   1.0      Alabaster (1969)

Table 6.  (contd.)
---------------------------------------------------------------------------------------------------------
Organism                 Flow/ Temp Alkali- Hard- pH   Formulationc   Parameter   Water      Reference
                         stata (°C) nityb   nessb                              concentration
                                                                                (mg/litre)
---------------------------------------------------------------------------------------------------------

Rainbow trout            flow  20            250  7.2  clay-based     24-h LC50   7000     Alabaster (1969)
 (Salmo gairdneri)                                      pellets
                         flow  20            250  7.2  clay-based     48-h LC50   4800     Alabaster (1969)
                                                       pellets
                         flow  20            250  7.2  resin-based    24-h LC50   3400     Alabaster (1969)
                                                       pellets
                         flow  20            250  7.2  resin-based    48-h LC50   2400     Alabaster (1969)
                                                       pellets
                         flow  20            250  7.2  amine salt     24-h LC50   250      Alabaster (1969)
                         flow  20            250  7.2  amine salt     48-h LC50   210      Alabaster (1969)
---------------------------------------------------------------------------------------------------------
a  Stat = static conditions (water unchanged for duration of test); 
   flow = flow-through conditions (2,4-D concentration in water 
   continuously maintained). 
b  Alkalinity & hardness expressed as mg CaCO3/litre. 
c  di-N,N = di-N,N-dimethylcocoamine; PGBEE = propylene glycol 
   butyl ether ester; total 2,4-D = the effect actually observed in the 
   flow-through test; the value which preceeds each "total 2,4-D" value is 
   the calculated effect of the ester alone.  The authors determined the 
   degree of hydrolysis of the ester during the course of the test and 
   subtracted the effect due to the free acid produced by this hydrolysis. 
6.2.1.1  Tolerance and potentiation

    Chambers  et al. (1977) used  insecticide-tolerant and insecticide-
susceptible populations of mosquito fish and an esterase  inhibitor  to
investigate  hydrolytic activation and detoxification  of 2,4-D esters.
Mosquito  fish taken from  a wild population  which had developed  some
tolerance  to insecticides also showed  some slight tolerance to  2,4-D
ethyl  and butyl esters.  This  tolerance was most pronounced  with the
butyl  ester, where the 48-h LC50 was  raised from 0.98 mg/litre in the
susceptible  fish, to  1.70 mg/litre  in the  tolerant  fish.   Further
experiments were carried out to find the basis for this  tolerance  and
for  the higher toxicity of  2,4-D esters over that  of the free  acid.
The  addition of DEF  (S,S,S-tributyl phosphorotrithioate), a  carboxyl
esterase  inhibitor, to the toxicity  test medium slightly reduced  the
toxicity of both 2,4-D esters.  This finding suggested that  the  toxic
effect  of  the esters  required initial hydrolysis  to the acid.   The
increased  toxicity of the esters  would result from esters  being more
readily  absorbed into the fish  through the gills.  The  resistance of
the  insecticide-tolerant population was, at least partially, explained
by  measuring esterase activity in  homogenates of gill and  liver from
the  two fish populations.  The  tolerant fish hydrolyzed less  of both
2,4-D esters than susceptible fish.  This effect was most  marked  with
liver homogenates; results from gill homogenates were  equivocal.   The
overall  conclusion,  put  forward  by  the  authors,  is  that   liver
hydrolysis  `activates' 2,4-D esters  by converting them  to the  toxic
free  acid.  Hydrolysis  in the  gill is  a  `detoxification'  reaction
because it reduces the uptake of toxic material.  The  slight  increase
in  tolerance in the insecticide-resistant mosquito fish is largely the
result  of  decreased  activation of  the  2,4-D  esters by  the liver.
Antagonism  to  2,4-D ester  toxicity by DEF  is largely the  result of
inhibition   of  activation  in   the  liver  rather   than   increased
detoxification  in  either  liver or  gill  (Chambers  et  al.,  1977).
Carbaryl, a cholinesterase inhibitor, potentiates the toxicity of 2,4-D
butyl  ester to brown  trout  (Salmo trutta)  (Statham  &  Lech,  1975).
The  4.5-h LC50 for  2,4-D butyl ester in static tests was shifted from
30  mg/litre  to  11  mg/litre  by  the  addition  of  carbaryl,  at  a
concentration  of  1  mg/litre, to  the  test  water.  Carbaryl  has no
toxicity to the fish at this concentration; the 24-h LC50 for  carbaryl
alone  is 6.8 mg/litre.  The potentiating effect of carbaryl was itself
blocked  by atropine, a muscarinic blocker, at a water concentration of
10  mg/litre;  the atropine  itself was not  toxic to the  fish at this
concentration.   In a similar way, carbaryl potentiated the toxicity of
several  other compounds.  The authors suggested a non-specific action,
possibly by increasing the uptake of 2,4-D ester from the  water.   The
same  potentiation  was demonstrated  for  trout in  flow-through tests
(Statham  &  Lech,  1975).  Combinations  of  2,4-D  esters  (butyl  or
propylene  glycol  butyl) with  the  herbicide picloram  increased  the
toxicity  to  fish  above  the  combined  toxicity  of  the  individual
compounds (Woodward, 1982).

6.2.2.  No-observed-effect levels in short-term tests with fish

    The  NOELs of 2,4-D on  fish in short-term tests  are summarized in
Table  7.   Values quoted  from Meehan et  al. (1974)  and  from Butler
(1965)  are based on the lowest dose used in their studies.  The values
from  Birge et al.  (1979) are calculated  1% mortality values  derived
mathematically  from  a full  toxicity curve, and  based on young  fish
exposed  to 2,4-D from  shortly after fertilization  of the eggs  until
4  days  after hatching.   The differing exposure  times for the  three
species  tested  is  due  to  differences  in  time to  hatching.   The
variation   in   these  data  is,  therefore,  partly  due  to  species
differences in sensitivity to the compound and partly due  to  exposure
times.   Newly hatched  young fish  are more  sensitive to  2,4-D  than
unhatched embryos (see section 6.2.4).

6.2.3.  Species differences in short-term toxicity to fish

    Variation  in  the  toxicity  of  2,4-D  to  fish with  species  is
summarized  in Table 8.  Of  a range of fish  species, examined in  the
same test conditions, using 2,4-D as the free acid, by Rehwoldt  et al.
(1977),  the  most sensitive  was  the white  perch (Roccus americanus)
with   a  24 h  LC50 of  55 mg/litre,  and the least  sensitive was the
eel  Anguilla rostrata  with an LC50 of 427 mg/litre.  The  grass  carp,
often used together with herbicides to control aquatic  vegetation, was
the least sensitive of all species examined, with a 24 h  LC50 for   an
amine salt formulation of 3080 mg/litre (Tooby et al., 1980).

6.2.4.  Toxicity to early life-stages of fish

    Short-term studies on the toxicity of 2,4-D to early life-stages of
fish are summarized in Table 9.

    Studies  on fish eggs  and larvae immediately  after hatching  have
been  conducted on few species  and mainly with simple  salts of 2,4-D.
There  is little information on  the effects of the  more toxic esters.
2,4-D  is clearly toxic for  fish early life-stages, within  the likely
range  of water concentrations  which would be  found after use  of the
herbicide to control aquatic weeds.

    At the 16 - 32 cell blastomere stage, eggs of the  bleak    Alburnus
 alburnus, developed more slowly than control eggs when exposed to 2,4-D
solutions.    After 48-h  exposure, mortality  reached 68%  and 79%  in
those  eggs  exposed  to 2,4-D  at  25  and 50  mg/litre, respectively.
Control  mortality  after  48-h  exposure  was  37%.   After  23  h  of
development,   eggs  exposed  to   25  mg  2,4-D/litre   showed  normal
development  while  those  exposed  to  100  mg/litre   showed   slower
embryogenesis  or  development  halted at  the  morula-gastrula  stage.
Free-swimming larvae were more sensitive to 2,4-D than eggs;  the  rate
of  survival of embryos in  tests lasting for between  12 and 48 h  was
higher than for larvae.  In tests lasting for between 24 and 48  h,  at
concentrations  of  2,4-D  above  400  mg/litre,  no  larvae  survived.
Embryos  showed  malformations  and reduced  mobility at concentrations
above  100  mg/litre, and  at concentrations of  800 mg/litre or  more,
embryos were immobile (Biro, 1979).

    Birge et al. (1979) examined the effects of 2,4-D, as the potassium
salt, on the eggs and larvae of three species of fish.   Rainbow  trout
eggs  were the most sensitive, largemouth bass eggs less sensitive, and
goldfish  eggs extremely  tolerant to  2,4-D.  In  all species  tested,
the  larval stages were more  sensitive to the herbicide  than were the
eggs.


    Table 7.  Toxicity of 2,4-D to fish: no-observed-effect levels
---------------------------------------------------------------------------------------------------------
Organism                 Flow/ Temp  Alkali- Hard-  pH    Formulationc  Parameter    Water      Reference
                         stata (°C)  nityb   nessb                                concentration
                                                                                   (mg/litre)
---------------------------------------------------------------------------------------------------------
Pink salmon (fry)        stat  10            10-34        free acid     96-h LC0     < 1       Meehan
 (Oncorhynchus            stat  10            10-34        butyl ester   96-h LC0     < 1       et al.
  gorbuscha               stat  10            10-34        isooctyl      96-h LC0     < 1       (1974)

Chum salmon (fry)        stat  10            10-34        free acid     96-h LC0     10         Meehan
 (Oncorhynchus keto)      stat  10            10-34        butyl ester   96-h LC0     < 1       et al.
                         stat  10            10-34        isooctyl      96-h LC0     1          (1974)

Coho salmon (fry)        stat  10            10-34        free acid     96-h LC0     10         Meehan
 (Oncorhynchus kisutch)   stat  10            10-34        butyl ester   96-h LC0     < 1       et al.
                         stat  10            10-34        isooctyl      96-h LC0     1          (1974)

Sockeye salmon (smolts)  stat  10            10-34        free acid     96-h LC0     10         Meehan
 (Oncorhynchus nerka)     stat  10            10-34        butyl ester   96-h LC0     < 1       et al.
                         stat  10            10-34        isooctyl      96-h LC0     < 1       (1974)

Alaska coho salmon       stat  10            10-34        free acid     96-h LC0     50         Meehan
     (fingerlings)       stat  10            10-34        butyl ester   96-h LC0     < 1       et al. 
 (Oncorhynchus kisutch)   stat  10            10-34        isooctyl      96-h LC0     < 1       (1974)
                         stat  10            10-34        PGBEE         96-h LC0     < 1       

Oregon coho salmon       stat  10            10-34        free acid     96-h LC0     10         Meehan
     (fingerlings)       stat  10            10-34        butyl ester   96-h LC0     < 1       et al.
 (Oncorhynchus kisutch)   stat  10            10-34        isooctyl      96-h LC0     10         (1974)

Dolly Varden             stat  10            10-34        free acid     96-h LC0     50         Meehan
     (fingerling)        stat  10            10-34        butyl ester   96-h LC0     < 1       et al.
 (Salvelinus malma)       stat  10            10-34        isooctyl      96-h LC0     10         (1974)

Rainbow trout            stat  10            10-34        free acid     96-h LC0     50         Meehan
     (fingerling)        stat  10            10-34        butyl ester   96-h LC0     < 1       et al. 
 (Salmo gairdneri)                                                                               (1974)

Spot                     stat                             free acid     48-h LC0     50         Butler 
 (Leistomus xanthurus)                                                                           (1965)

Longnose killifish       stat                             dimethylamine 48-h LC0     15         Butler 
 (Fundulus similis)                                                                              (1965)
---------------------------------------------------------------------------------------------------------

    Table 7.  (contd.)
---------------------------------------------------------------------------------------------------------
Organism                 Flow/ Temp  Alkali- Hard-  pH    Formulationc  Parameter    Water      Reference
                         stata (°C)  nityb   nessb                                concentration
                                                                                   (mg/litre)
---------------------------------------------------------------------------------------------------------
Mullet (Mugil cephalus)  stat                             ethylhexyl    48-h LC0     10         Butler 
                                                                                                (1965)
White mullet (juvenile)  flow         sea                 free acid     48-h LC0     50.0       Butler 
 (Mugil curema)                        water                                                     (1963)

Goldfish                 flow  18.2-  66.7   53.3   7.84  pot. salt     8-day LC1    8.2        Birge et al.
 (Carassius auratus)            25.8                                               (2.7-15.0)    (1979)d
                         flow  18.2-  65.3   197.5  7.78  pot. salt     8-day LC1    8.9        Birge et al.
                               25.8                                               (3.8-14.6)    (1979)d

Largemouth bass          flow  18.2-  66.7   53.5   7.84  pot. salt     7.5-day LC1  13.1       Birge et al.
 (Micropterus salmoides)        25.8                                               (4.4-21-9)    (1979)d
                         flow  18.2-  65.3   197.5  7.78  pot. salt     7.5-day LC1  3.2        Birge et al.
                               25.8                                               (1.2-6.0)     (1979)d

Rainbow trout            flow  12.5-  66.7   53.5   7.84  pot. salt     27-day LC1   0.032      Birge et al.
 (Salmo gairdneri)              14.5                                               (0.008-0.084) (1979)d
                         flow  12.5-  65.3   197.5  7.78  pot. salt     27-day LC1   0.022      Birge et al.
                               14.5                                               (0.006-0.055) (1979)d
---------------------------------------------------------------------------------------------------------
a  Stat = static conditions (water unchanged for duration of test); flow = flow-through conditions 
   (2,4-D concentration in water continuously maintained).
b  Alkalinity & hardness expressed as mg CaCO3/litre.
c  pot. salt = potassium salt; PGBEE = propylene glycol butyl ether ester.
   LC0 obtained by extrapolation and LC1 mathematically calculated.
d  Birge et al. (1979) exposed fish from four days after hatching.

Table 8.  Toxicity of 2,4-D to fish: species variation
---------------------------------------------------------------------------------------------------------
Organism                  Flow/ Temp Alkali- Hard-  pH   Formulationc  Parameter     Water     Reference
                          stata (°C) nityb   nessb                                concentration
                                                                                   (mg/litre)
---------------------------------------------------------------------------------------------------------
Striped bass              stat  20           50     7.2  free acid     24-h LC50     85.6      Rehwoldt
 (Morone saxatilis)        stat  20           50     7.2  free acid     96-h LC50     70.1      et al. 
                                                                                               (1977)
Banded killifish          stat  20           50     7.2  free acid     24-h LC50     306.2     Rehwoldt
 (Fundulus diaphanus)      stat  20           50     7.2  free acid     96-h LC50     26.7      et al. 
                                                                                               (1977)
Pumpkinseed sunfish       stat  20           50     7.2  free acid     24-h LC50     120       Rehwoldt
 (Lepomis gibbosus)        stat  20           50     7.2  free acid     96-h LC50     94.6      et al. 
                                                                                               (1977)
White perch               stat  20           50     7.2  free acid     24-h LC50     55.5      Rehwoldt
 (Roccus americanus)       stat  20           50     7.2  free acid     96-h LC50     40        et al. 
                                                                                               (1977)
American eel              stat  20           50     7.2  free acid     24-h LC50     427.2     Rehwoldt
 (Anguilla rostrata)       stat  20           50     7.2  free acid     96-h LC50     300.6     et al. 
                                                                                               (1977)
Carp                      stat  20           50     7.2  free acid     24-h LC50     175.2     Rehwoldt
 (Cyprinus carpio)         stat  20           50     7.2  free acid     96-h LC50     96.5      et al. 
                                                                                               (1977)
Guppy                     stat  20           50     7.2  free acid     24-h LC50     76.7      Rehwoldt
 (Lebistes reticulata)     stat  20           50     7.2  free acid     96-h LC50     70.7      et al. 
                                                                                               (1977)
Grass carp                flow  13           270    8.1  amine salt    24-h LC50     3080      Tooby et 
 (Ctenopharyngodon                                                                 (2622-3618)  al. (1980)
  idella)                  flow  13           270    8.1  amine salt    48-h LC50     2540      Tooby et 
                                                                                  (2184-2952)  al. (1980)
                          flow  13           270    8.1  amine salt    96-h LC50     1313      Tooby et 
                                                                                  (1116-1544)  al. (1980)

Bleak                     stat  10   15             7.8  butoxyethanol 96-h LC50    3.2-3.7    Linden et 
 (Alburnus alburnus)                                                                            al. (1979)

Mosquito fish             stat  21-22                    amine salt    24-h LC50     500       Johnson 
 (Gambusia affinis)        stat  21-22                    amine salt    48-h LC50     445       (1978)
                          stat  21-22                    amine salt    96-h LC50     405       

Mullet                    stat                           sodium salt   24-h LC50     68.0      Tag El-Din
 (Mugil cephalus)          stat                           sodium salt   96-h LC50     32.0      et al.
                                                                                               (1981)
Longnosed killifish       stat                           butoxyethanol 48-h LC50     5.0       Butler 
 (Fundulus similis)        stat                           PGBEE         48-h LC50     4.5       (1965)
---------------------------------------------------------------------------------------------------------

Table 8.  (contd.)
---------------------------------------------------------------------------------------------------------
Organism                  Flow/ Temp Alkali- Hard-  pH   Formulationc  Parameter     Water     Reference
                          stata (°C) nityb   nessb                                concentration
                                                                                   (mg/litre)
---------------------------------------------------------------------------------------------------------

Bluegill sunfish          stat  25           19     7.0  dimethylamine 24-h LC50     390       Davis &
 (Lepomis macrochirus)     stat  25           19     7.0  dimethylamine 48-h LC50     375       Hardcastle
                                                                                               (1959)

Largemouth bass           stat  25           19     7.0  dimethylamine 24-h LC50     375       Davis &
 (Micropterus salmoides)   stat  25           19     7.0  dimethylamine 48-h LC50     350       Hardcastle
                                                                                               (1959)
Punti  (Puntius ticto)     stat  23.5                     ethyl ester   24-h LC50     1.6       Verma et 
                                                                                               al. (1984)
Medaka  (Oryzias latipes)                                 sodium salt   48-h LC50     > 40      Hashimoto &
                                                                                               Nishiuchi
                                                                                               (1978)

Longnose killifish (juv.) flow       sea water           PGBEE         24-h LC50     5.0       Butler 
 (Fundulus similis)        flow       sea water           PGBEE         48-h LC50     4.5       (1963)
                          flow       sea water           butoxyethanol 24-h LC50     5.0       Butler 
                          flow       sea water           butoxyethanol 48-h LC50     5.0       (1963)
---------------------------------------------------------------------------------------------------------
a  Stat = static conditions (water unchanged for duration of test); flow = flow-through conditions 
   (2,4-D concentration in water continuously maintained).
b  Alkalinity & hardness expressed as mg CaCO3/litre.
c  PGBEE = propylene glycol butyl ether ester

Table 9.  Toxicity of 2,4-D to fish early life-stages
---------------------------------------------------------------------------------------------------------
Organism                 Flow/  Temp Alkali- Hard-  pH   Formulation  Parameter    Water       Reference
                         stata  (°C) nityb   nessb                              concentration
                                                                                 (mg/litre)
---------------------------------------------------------------------------------------------------------
Bleak      (embryo)                                      sodium salt  12-h LC50    159.4       Biro (1979)
 (Alburnus alburnus)                                      sodium salt  24-h LC50    129.0       Biro (1979)
                                                         sodium salt  36-h LC50     63.9       Biro (1979)
                                                         sodium salt  48-h LC50     12.9       Biro (1979)

           (larvae)                                      sodium salt  12-h LC50    111.2       Biro (1979)
                                                         sodium salt  24-h LC50     70.6       Biro (1979)
                                                         sodium salt  36-h LC50     62.1       Biro (1979)
                                                         sodium salt  48-h LC50     51.6       Biro (1979)

Goldfish   (embryo)      flow  18.2-  66.7   53.3   7.84 pot. salt    4-day LC50   > 187       Birge et 
 (Carassius auratus)            25.8                                                            al. (1979)
                         flow  18.2-  65.3   197.5  7.78 pot. salt    4-day LC50   > 201       Birge et 
                               25.8                                                            al. (1979)
           (4-day        flow  18.2-  66.7   53.3   7.84 pot. salt    8-day LC50   133.1       Birge et 
         post-hatch)           25.8                                             (108.6-174.8)  al. (1979)
                         flow  18.2-  65.3   197.5  7.78 pot. salt    8-day LC50   119.1       Birge et 
                               25.8                                             (98.5-150.6)   al. (1979)

Largemouth bass (embryo) flow  18.2-  66.7   53.3   7.84 pot. salt    3.5-day LC50 165.4       Birge et 
 (Micropterus salmoides)        25.8                                             (130.6-274.1)  al. (1979)
                         flow  18.2-  65.3   197.5  7.78 pot. salt    3.5-day LC50 160.7       Birge et 
                               25.8                                             (122.9-230.6)  al. (1979)
           (4-day        flow  18.2-  66.7   53.3   7.84 pot. salt    7.5-day LC50 108.6       Birge et 
         post-hatch)           25.8                                             (92.5-138.4)   al. (1979)
                         flow  18.2-  65.3   197.5  7.78 pot. salt    7.5-day LC50  81.6       Birge et 
                               25.8                                             (64.8-103.5)   al. (1979)

Rainbow trout (embryo)   flow  18.2-  66.7   53.3   7.84 pot. salt    23-day LC50   11.0       Birge et 
 (Salmo gairdneri)              25.8                                              (7.8-15.1)    al. (1979)
                         flow  18.2-  65.3   197.5  7.78 pot. salt    23-day LC50    4.2       Birge et 
                               25.8                                               (2.8-5.9)    al. (1979)
           (4-day        flow  18.2-  66.7   53.3   7.84 pot. salt    27-day LC50   11.0       Birge et 
        post-hatch)            25.8                                              (7.8-15.1)    al. (1979)
                         flow  18.2-  65.3   197.5  7.78 pot. salt    27-day LC50    4.2       Birge et 
                               25.8                                               (2.8-5.9)    al. (1979)
---------------------------------------------------------------------------------------------------------
a  Stat = static conditions (water unchanged for duration of test); flow = flow-through conditions (2,4-D concentration
   in water continuously maintained).
b  Alkalinity & hardness expressed as mg CaCO3/litre.
   Pot. salt = potassium salt.
    Only one study has examined the effects of 2,4-D esters  on  newly-
hatched fish fry and on fertilized eggs.  Unfortunately, this study, by
Hiltibran  (1967), does not record the full experimental details.  Four
species  of fish were used in the study but complete results were given
only  for the bluegill sunfish.   The most toxic preparations  were the
propylene glycol butyl ether (PGBE) ester and mixed isopropyl and butyl
esters   with   no-observed-effect  levels   of   2  and   3  mg/litre,
respectively.   The  dimethylamine  salt, ethylhexyl  ester, and sodium
salt formulations were less toxic with no-observed-effect levels at 40,
50, and 100 mg/litre, respectively.

6.2.5.  Long-term toxicity to fish

    Chronic exposure of sub-adult fish of a variety of species to 2,4-D
for  10 months, at a concentration of 0.1 mg/litre, led to no mortality
and to no change in the acute response to 2,4-D.  The 24-h  LC50    for
the  compound was  unchanged after  10 months  exposure  to  sub-lethal
doses;  i.e., no tolerance developed.   One species of fish,  the guppy
 Lebistes  reticulatus,  reproduced in captivity.   Reproductive success
was   compared   between  control  fish  and  fish  breeding  in  water
containing  2,4-D at 0.1 mg/litre over 10 months.  The ratio of numbers
of  offspring of treated  and control fish  was 1.2  (Rehwoldt  et al.,
1977).   Mount & Stephan (1967)  conducted a 10-month study  on fathead
minnows  (Pimephales promelas)  exposed  to  the butoxyethanol  ester of
2,4-D  at  0,  0.01, 0.04, 0.2, or 0.8 mg/litre water in a flow-through
test  system.  Concentrations of 2,4-D  of 0.2 mg/litre or  less had no
observable effect on growth, survival, or reproductive success  of  the
fish,  but  the highest  concentration tested was  toxic to eggs.   The
highest   tested   no-observed-effect  concentration was  approximately
1/45  of the 96-h  LC50 for  this species.   Finlayson & Verrue  (1985)
conducted   a  chronic   egg-to-fry test  over 86  days  using  chinook
salmon   in  2,4-D  butoxyethanol   ester  solutions  ranging   up   to
118 µg/litre.   The mortality of salmon during the alevin to fry period
was 4.7% and 47.6% for exposures to 60 and 118 µg/litre,  respectively.
When   compared  to  controls,   the  length  of   salmon  at  36  days
post  hatch  was  significantly  reduced  after  exposure  to  60   and
118 µg/litre.    Neither survival nor  growth of fry  were affected  at
2,4-D  concentrations of 40 µg/litre  or less.  This maximum acceptable
toxicant  concentration (MATC)  represents 0.13  and 0.11  of the  96-h
LC50 values for fry and  smolts of this species, respectively.

6.2.6.  Behavioural effects on fish

    Folmar  (1976)  used  rainbow trout  fry  in  an  investigation  to
determine whether fish avoided water contaminated by herbicides.  Trout
fry  were  initially  placed in  a  `Y'-shaped  maze for  15  min.  The
dimethylamine  salt of 2,4-D, then was added to one arm of the maze and
clean  water in a second  arm, both at flow  rates of 400 ml/min.   The
number  of fish in each arm was counted after 1 hour and results tested
statistically  using a Chi-squared test.  At concentrations of 2,4-D of
0.1 mg/litre water (approximately equal to water levels after  the  use
of this preparation as an aquatic herbicide), there was  no   avoidance
of  the compound; the  numbers of fish  in each arm  of the  maze  were
equal.   However, at concentrations of  1.0 or 10.0 mg/litre  of 2,4-D,
there was significant avoidance of the chemical.

    Hidaka  et  al.  (1984) conducted  a  similar  study using  medakas
 Oryzias   latipes,   and  tested a wide range of doses for a variety of
pesticides   and  herbicides.   In all  cases,  the  fish  avoided  the
chemical  in a dose-related  manner, but only  over a limited  range of
concentrations.  Above this range, presumably because of the  onset  of
toxic effects, there was a dose-related decrease in avoidance response.
The  authors  calculated  two values from these chevron-shaped  graphs,
the   avoidance   response  EC65 taken   from  the  increasing    curve
(AR65)   and the DAR60 taken  from the decreasing curve. For 2,4-D, the
value for AR65 was  177 (171-182; 95% confidence limits) µg/litre,  and
for  DAR60 was  288 (245-338) µg/litre.    Compared to other  chemicals
commonly  used  or  found in  water,  2,4-D  has a  high  threshold  of
detection by fish as indicated by the high avoidance threshold.

    Rand & Barthalmus (1980) exposed goldfish to 2,4-D at  20  mg/litre
(10% of the 96-h LC50 for  this species) at different stages during the
training  period for conditioning  the fish to  avoid electric  shocks.
They  found that the herbicide had no effect when given for 24 h on the
9th  day of conditioning but was effective in changing the magnitude of
the avoidance response when given for the first 24 h  of  conditioning.
Fish exposed for 2 weeks showed significant differences in the pattern,
rate  of  acquisition,  and  maintenance  of  the  avoidance  baseline.
Behavioural differences persisted into the post-exposure period in fish
exposed  to 2,4-D for 2  weeks.  The authors point  out that short-term
toxicity tests do not examine subtle behavioural effects which could be
of considerable importance in the wild.

    Dodson  &  Mayfield  (1979) assessed  the  effect  of 2,4-D  on the
``reotaxic  response'' of rainbow trout, that is their tendency to swim
upstream  to compensate for  flowing water.  There  was a  dose-related
effect of 2,4-D at concentrations between 0 and 7 mg/litre.  Above this
concentration range, there was a fall of more than 50% in the frequency
of positive reotaxic response to a revolving drum marked  in  alternate
light  and  dark  stripes and  an  increase  in the  frequency  of `no-
response'.   The authors state that, at ``realistic concentrations'' of
2,4-D  in  water,  there  would  be a  tendency  for fish  to  be moved
downstream because of a reduced reotaxic response.

6.2.7.  Effects of environmental variables on toxicity to fish

    The   toxicity  of 2,4-D  to fish is  related to season.   Vardia &
Durve  (1981) obtained  different values  for 96-h  LC50 for  the  carp
 Cyprinus    carpio  at   different   times   of    the   year.    Water
characteristics  did not differ,  except for temperature  which  varied
from 39 °C in May, its highest value, to 17 °C in February, its lowest.
There  was a positive correlation between temperature and toxicity.  At
39 °C, the 96-h LC50 was  5.6 mg/litre, and, at 17 °C, the  LC50    was
40.83 mg/litre.  As the authors point out, the temperature of the water
must  be borne in mind  when assessing the safety  of this compound  to
fish  during control of aquatic weeds.  The effect may be one of season
rather  than  temperature;  the physiology  of  the  fish also  changes
throughout the year.

    There  is some effect of water hardness and pH on 2,4-D toxicity to
fish but this is very dependent on the species of test fish  (Birge  et
al., 1979).  This effect has not been studied systematically.

6.2.8.  Special studies on fish

    Chronic  exposure  of  carp to sub-lethal concentrations  of  2,4-D
(5  mg/litre) led to ultrastructural  changes in the liver  of the fish
(Benedeczky  et  al.,  1984).  After  2  months,  there was  detectable
swelling  of mitochondria and loss  of cristae.  There were  also large
numbers of inclusions in the cytoplasm, interpreted by the  authors  as
bile  pigments.  Their presence  in the bile  canaliculi indicated  the
onset of cholestasis (reduction in bile flow).  After 3, 4, or 5 months
of exposure, the cholestasis was pronounced with  cholesterin  crystals
appearing  as  cytoplasmic  inclusions.  Later,  in  the  6th month  of
exposure,  there  were  endoplasmic  reticulum  changes  indicative  of
changed protein synthesis.

    Oxygen consumption by bluegill sunfish was not affected by 2,4-D at
a concentration of 3 mg/litre water (Sigmon, 1979).

    2,4-D  at 10-4   mol/litre of medium did not affect the activity of
Na/K-ATPase in microsomes from trout gill (Davis et al., 1972).

    Verma  et  al.  (1984) detected  effects  on  pituitary and  pineal
histology  in punti  Puntius ticto  exposed  for 96 h  to 1 mg/litre  of
Weedone (ethyl ester of 2,4-D).  There was a significant effect on cell
size  of acidophilic pituitary cells  but a much more  marked effect on
cyanophils.  Pineal epithelium cell height was significantly greater in
exposed fish.

6.3.  Toxicity to Amphibians

 Appraisal

     Amphibian  larvae are generally tolerant  to amine salts of  2,4-D;
 the 96-h  LC50  values exceed 100 mg/litre.  Of the species tested, only
 one was sensitive.

     No  information  is  available  on  reproductive  development   and
 differentiation or on tissue levels.

    The  toxicity  of 2,4-D  to amphibians is  summarized in Table  10.
Tadpoles  of  the  Indian toad  are  particularly  susceptible  to  the
compound   (Vardia  et al.,  1984).  Lhoste &  Roth (1946) showed  that
2,4-D,  at 5 g/litre, prevented  development of the eggs  of the common
frog (Rana temporaria).  At doses between 500 mg/litre and  4  g/litre,
there was some development which decreased with increasing dose.  These
levels  are  far  higher than  those  likely  to be  encountered in the
environment.


Table 10.  Toxicity of 2,4-D to amphibians
---------------------------------------------------------------------------------------------------------
Organism                 Flow/  Temp  Alkali- Hard-  pH   Formulation    Parameter     Water      Refer-
                         stata  (°C)  nityb   nessb                                 concentration ence
                                                                                     (mg/litre)
-------------------------------------------------------------------------------------------------------------------------------------------------
Chorus frog (tadpole)    stat   15.5  30             7.1  dimethylamine  24-h LC50     > 100     Sanders
 (Pseudacris triseriata)  stat   15.5  30             7.1  dimethylamine  96-h LC50     > 100     (1970b)

Indian toad (tadpole)           25    210     220    8.3  free acid      24-h LC50      13.77     Vardia et 
                                                                                    (11.81-16.05) al. (1984)
 (Bufo melanostictus)            25    210     220    8.3  free acid      48-h LC50       9.03     Vardia et 
                                                                                     (8.23-9.91)  al. (1984)
                                25    210     220    8.3  free acid      96-h LC50       8.05     Vardia et 
                                                                                     (7.29-8.81)  al. (1984)

Frog (tadpole)           stat   21-22                     amine salt     24-h LC50     255        Johnson 
 (Adelotus brevis)        stat   21-22                     amine salt     48-h LC50     228        (1976)
                         stat   21-22                     amine salt     96-h LC50     200        Johnson 
                                                                                                  (1976)
Frog (tadpole)           stat   21-22                     amine salt     24-h LC50     321        Johnson 
 (Limnodynastes peroni)   stat   21-22                     amine salt     48-h LC50     300        (1976)
                         stat   21-22                     amine salt     96-h LC50     287        Johnson 
                                                                                                  (1976)
Toad (tadpole)           stat   21-22                     amine salt     24-h LC50     346        Johnson 
 (Bufo marinus)           stat   21-22                     amine salt     48-h LC50     333        (1976)
                         stat   21-22                     amine salt     96-h LC50     288        Johnson 
                                                                                                  (1976)
Common frog (tadpole)           17-29                     free acid      48-h LC0       50        Cooke 
 (Rana temporaria)                                                                                 (1972)
-------------------------------------------------------------------------------------------------------------------------------------------------
a  Stat = static conditions (water unchanged for duration of test); flow = flow-through conditions 
   (2,4-D concentration in water continuously maintained).
b  Alkalinity and hardness expressed as mg CaCO3/litre.
7.  TOXICITY TO TERRESTRIAL ORGANISMS

 Appraisal

     For terrestrial application, 2,4-D is usually used in the  form  of
 the less volatile, longer-chain esters to reduce drift damage of sprays
 to broad-leaved crop plants.  The herbicide is used on cereal crops and
 on  rangeland against broad-leaved weeds  and also in forestry.   Thus,
 insects of all kinds will be exposed to 2,4-D.  Birds' eggs in the nest
 are  more  likely  to be  exposed  than  the adult  birds, though adult
 exposure  remains  possible,  particularly for  sitting birds.  Ground-
 nesting  species will be exposed from the use of 2,4-D on rangeland and
 cereals; tree-nesting species will be exposed in forests.

     The  insecticidal action of 2,4-D  is low enough that  the compound
 does not represent a hazard to beneficial insects; there is an adequate
 safety margin with usage at recommended levels.

     Although  there is some  disagreement in the  literature about  the
 toxicity  of  2,4-D  to birds'  eggs,  the  low uptake  of the material
 through  the egg shell suggests that exposure would not affect hatching
 in  normal use of the compound.  Adult birds are not affected by short-
 term  exposure  to  2,4-D.  The  likelihood  of  prolonged exposure  of
 either adult birds or eggs to high levels of 2,4-D is small.

7.1.  Toxicity to Terrestrial Invertebrates

 Appraisal

     Based  on the widespread use of 2,4-D and its formulations, insects
 of many kinds could be exposed to the material.  Although the compounds
 are generally classified as non-toxic for beneficial insects,  such  as
 honey bees and natural enemies of pests, some adverse effects have been
 reported on the early life-stages and adults of some insects.

    Esters are less toxic to insects than are salts or the free acid.

    Feeding   studies dosing worker  honey bees (Apis  mellifera)  with
2,4-D  salts in  sucrose syrup  have generated  two estimates  of  24-h
LC50:     104 and 115 µg/bee  (Jones & Connell, 1954; Beran & Neururer,
1955).   Morton  et  al. (1972)  fed 2,4-D  acid to  honey bees  in 60%
sucrose  syrup at 10,  100, or 1000  mg/litre and monitored  half-time,
i.e., the time taken for 50% of the bees in a cage to die.   The  half-
time  was significantly longer than that of controls for the two lowest
doses   (37.2  days  at 10  mg/litre  and  40.4 days  at  100 mg/litre,
compared  to  a  control value  of  33.4  days), but  was significantly
reduced at 1000 mg/litre (18.6 days).  The butoxyethanol  and  isooctyl
(commercial  formulation) esters  of 2,4-D  had no  effect on  survival
times  at 10, 100, or  1000 mg/litre of syrup  when fed under the  same
conditions  as the acid.  The  dimethylamine salt of 2,4-D  (commercial
formulation) had no effect at 10 or 100 mg/litre, but did  shorten  the
half-time at 1000 mg/litre.

    The  effects of 2,4-D on beneficial coccinellid larvae were studied
by Adams (1960).  Larvae were sprayed with a preparation of mixed amine
salts  of 2,4-D, at a rate equivalent to 0.56 kg acid equivalent/ha, at
different  stages  of their  development 1, 3,  6, 9, or  12 days after
hatching.  There was a lengthening of the development period  when  the
larvae were treated on days 3, 6, 9, or 12 but no effect when they were
sprayed on the first day after hatching.  Mortality before pupation was
more  than doubled in all treated groups, but mortality during pupation
was not different from that of controls.

    Trumble   &   Kok   (1980)  dosed   adult  thistle-rosette  weevils
 (Ceuthorhynchidius   horridus),  which  are  used  for  the  biological
control  of  musk thistle,  with 2,4-D amine  salt at five  dose levels
between    0.17   and   147.8  kg/ha.   No  significant  mortality  was
observed,  up to 175 days after treatment, at doses up to and including
1.68  kg/ha.  At 16.8 and  84 kg/ha, there was  significantly increased
mortality  after day 3  post-treatment.  At the  highest dose level  of
147.8  kg/ha, mortality was increased  both on day 3  and subsequently.
Five-day LC50 values  for males and females were calculated at 70.2 and
61.4  kg/ha,  respectively.   This  is  41.8  times   the   recommended
application  rate  of 2,4-D  for males and  36.6 for females.   Riviere
(1976)  reared  the  European  cockroach  (Blatella germanica)  on  food
containing  1000 mg/kg and reported negligible effects on reproduction.

    Gall  & Dogger (1967) wetted  wheat plants with a  0.3% solution of
mixed  isopropyl and butyl  esters of 2,4-D  and exposed the  plants to
females  of the wheat stem  sawfly  (Cephus cinctus).   Spraying of  the
wheat  plants was performed at different times relative to oviposition;
times  were 7 days prior to oviposition, at the time of oviposition, or
7,  14, or 21 days after oviposition.  Eggs took about 7 days to hatch.
The  highest larval mortality  (96.4%) occurred after  spraying at  the
time of egg laying.  The effectiveness of the 2,4-D in  killing  larvae
decreased  with later exposure times.  For plants sprayed 7, 14, and 21
days  after oviposition, larval mortalities were 68.1%, 60.8%, and 37%,
respectively, compared to a control mortality of 30%.  When plants were
sprayed  7 days before egg  laying, larval mortality was  46.9%.  Adult
flies were not affected by 2,4-D spray.

    Muller   (1971)  exposed  beetles  (Carabidae) to sand  dose d  with
2,4-D   at   0.2,  1.0,   or  2.0  g/m2.      Two  species,    Bembidion
 femoratum  and  B. ustulatum, both showed more than 50% mortality within
4  days of  exposure to  1.0 g  2,4-D/m2.    B.  ustulatum  showed  100%
mortality  within  10  days when  exposed  to  1.0 g/m2    and  similar
mortality within 4 days when exposed to 2.0 g/m2.    About 20%  of  the
individuals  of  B. femoratum  survived the  14-day exposure to  both 1.0
and 2.0 g/m2.

    Roberts & Dorough (1984) exposed earthworms to 2,4-D  acid  sprayed
on  to filter paper.  The papers were wetted and the earthworms exposed
to the wetted paper in glass vials.  The calculated 48-h  LC50    value
was 61.6 (41 - 92.4; 95% confidence limits) µg/cm2.

    Rapoport  &  Cangioli  (1963)  treated  turf  with  a  mixture   of
(4-chloro-2-methylphenoxy)acetic acid (MCPA), at recommended rates, and
the  butyl ester  of 2,4-D,  at 10  times the  recommended rate.   They
reported no effect on soil microarthropods.

7.2.  Toxicity to Birds

 Appraisal

     Birds, and particularly the eggs of ground-nesting  species,  would
 be  exposed to 2,4-D after spraying.  Food items could also be expected
 to  be contaminated by the  herbicide.  However, most studies  on birds
 and their eggs have been conducted at exposures far higher  than  could
 be expected in the field.

     LD50 values   from acute oral  and from short-term  dietary  dosing
 indicate  low toxicity  of 2,4-D  to birds.   In  longer-term  studies,
 effects  have  only  been reported  at  extremely  high exposures  (for
 example,   kidney  effects  after   dosing  in  drinking   water   with
 concentrations  in excess of  the solubility of  the material).   There
 have  been no  reported effects  on reproductive  parameters,  even  at
 excessive exposure levels.

     A  single study reported adverse  effects on the embryos  of birds'
 eggs  sprayed with 2,4-D.  Many  studies since have shown  no effect on
 hatchability  of eggs and  no increased incidence  of abnormalities  in
 chicks  even after very high exposure to 2,4-D.  Other work indicates a
 very poor penetration of the eggshell by the herbicide.  It can only be
 concluded  that after normal, or even after excessive, 2,4-D use, there
 would be no effect on birds' eggs.

7.2.1.  Toxicity to birds' eggs

    There  have been several studies  on the toxicity of  various 2,4-D
formulations to birds' eggs dosed by different routes.

    Spraying eggs of pheasant, red-legged partridge, and grey partridge
with  the  equivalent  of  0.55  to  1.1  kg  2,4-D/ha,  either  before
incubation  or after 3 days of incubation, was found by Lutz-Ostertag &
Lutz  (1970)  and  Lutz  &  Lutz-Ostertag  (1972)  to  cause  embryonic
abnormalities.   They reported 77% mortality  in pheasant eggs, 77%  in
grey  partridge eggs, and 43%  in red-legged partridge eggs  within the
first 19 days of incubation.  The eggs were broken open,  between  days
20  and  22  of incubation  for  histopathological  examination of  the
embryos;  hatching takes place at about 24 days in all species used.  A
majority  of  surviving  embryos  were  either  wholly   or   partially
paralyzed.   Histopathological  effects  were mainly  gonadal.  In both
male and female embryos, there were abnormalities of the  gonad,  often
severe  enough  to  lead  to  sterility,  and,  in the  male,  abnormal
regression of the Mullerian ducts.  No control embryos  were  examined.
In  a second study by Lutz & Lutz-Ostertag (1973), quail, pheasant, and
partridge  eggs were sprayed with  2,4-D from two different  commercial
sources either before incubation or on days 3 or 7 after the  start  of

incubation.   The  authors  reported increased  mortality  in  embryos,
reduced  hatchability, and increased  abnormality in chicks.   The only
control  eggs reported  were those  in a  single incubation  of  quail.
Later work has failed to repeat these results.

    Kopischke  (1972) found no adverse  effects on hatchability and  no
increase  in deformities  or later  mortality of  hatched chicks  after
spraying  eggs  of  pheasants  or  chickens  with  an  isooctyl   ester
formulation  of  2,4-D  on day 13 of incubation at a dose equivalent to
0.28 kg/ha.

    Somers  et al. (1974)  sprayed chicken eggs,  prior to  incubation,
with  concentrations of an amine  salt of 2,4-D at  up to 15 times  the
recommended field application rate of approximately 3 kg/ha.  There was
no  effect on hatching  success or on  the survival of  chicks  in  the
period 3 to 4 weeks post hatch.  Spraying chicken eggs on days 0, 4, or
18 of incubation with 2,4-D (as a PGBE ester formulation) at up  to  10
times the field application rate, had no effect on hatchability  or  on
survival  and growth of chicks  after hatching (Somers et  al., 1978a).
Birds  hatched from  eggs, similarly  treated by  spraying,  showed  no
significant  adverse  effects  on later  reproductive  performance (egg
laying  performance  of the  females; testis weight  or sperm count  of
males) (Somers et al., 1978b).  Hilbig et al. (1976a) found  no  effect
on  egg hatch rate or  on body weight or  malformation rate in  chicks,
after spraying (at 20 kg/ha) the eggs of Japanese quail, pheasants, and
chickens  prior to incubation, or 3 days after the start of incubation.
In a follow-up study on the reproductive performance of  birds  hatched
from  these dosed eggs,  Hilbig et al.  (1976b) reported no  effects on
laying capacity, fertility, or hatchability of their eggs.

    The  effects of 2,4-D  dimethylamine salt on  the eggs of  Japanese
quail,  grey  partridge,  and  red-legged  partridge  were  studied  by
Grolleau  et al. (1974).  Eggs  were sprayed with 2,4-D  at dose levels
equivalent to the recommended application rate (1.2 kg/ha) and  at  two
higher  dose  levels  equivalent to  2.4  and  6 kg/ha.   There were no
effects  on hatching rate, embryonic  mortality, or chick mortality  in
the  first month after hatching or on embryonic or chick malformations.
In  addition, the histopathological  examination of partridge  thyroids
revealed  no  effects.   Residues  of  2,4-D  were  measured  in  those
partridge  eggs  receiving  the  highest  dose.   Very   little   2,4-D
penetrated the egg shell and the highest residue measured was  a  total
egg content of 19.3 µg  (in an 11-g egg, 15 days after treatment).  The
lack  of  effect of  2,4-D on sprayed  eggs was attributed  to the poor
penetration  of  the  herbicide.   Spittler  (1976)   found  no adverse
effects of 2,4-D on hatchability and no increase in chick abnormalities
in pheasant or quail eggs sprayed 24 h before hatching with a  dose  12
times  higher than the recommended application rate.  Only at a dose 30
times  higher than the recommended rate did hatchability fall by 10% to
15%, relative to controls.  No increased incidence of abnormalities was
reported at this dose rate.

    Hoffman  & Albers (1984)  immersed mallard eggs  for 30 seconds  in
aqueous  emulsions of  2,4-D and  calculated an  LC50 equivalent  to  a
field  application rate of 216 (155 - 300) kg/ha.  This is 32 times the
recommended  field  application  rate.   Dunachie  &  Fletcher   (1967)
injected chicken eggs with 10, 100, or 200 mg 2,4-D/kg,  equivalent  to
0.5,  5,  or  10 mg/egg,  and  found  reduced hatchability  relative to
control eggs injected with solvent only.  Treated eggs  showed  80-90%,
70%,  and  50%  of the  control hatch  rate for  the three  dose rates,
respectively.   In  a  similar study,  Gyrd-Hansen & Dalgaard-Mikkelsen
(1974)  found that injecting 1 mg/egg or less of the dimethylamine salt
of   2,4-D  had  no  effect.   Injections  of  2  mg/egg  reduced  both
hatchability of the eggs and survival of hatched chicks.   An  injected
dose of 5 mg/egg reduced the hatching rate to 15% of control levels and
there were no surviving chicks after 1 week.  There was  no  successful
hatching  after an injection of 10 mg/egg.  The same authors also dosed
eggs by immersion in solutions of 2,4-D for 10 seconds.  There  was  no
effect  after  immersion  in a solution of 10 g/litre and only a slight
effect  after  immersion  in 50  g/litre.   The  hatching  success  and
survival  of the chicks up  to 4 weeks post  hatch, after immersion  in
50 g/litre, was more than 80% of control values.

7.2.2.  Toxicity to birds after short-term and long-term dosing

    The  toxicity of 2,4-D  (given either orally  by capsule or  in the
diet) to birds is summarized in Table 11.  The studies reported in this
Table  include single oral  dosing, repeated oral  dosing, and  dietary
tests  over  5  to 100 days.  Studies lasting 10 days or less show that
high dosage (in excess of 1000 mg/kg food) is required to  kill  birds.
2,4-D is, therefore, of low toxicity to birds.

    Haegele & Tucker (1974) dosed egg-laying Japanese quail and mallard
a  single oral dose  of 250 or  1500 mg 2,4-D  acid/kg body weight  and
monitored  egg shell thickness.  There  was a short-term effect;  thin-
shelled  eggs were produced during the first 3 days after dosing.  This
was  considered to be an  indirect effect, i.e., the  result of reduced
food  consumption.  When Bjorklund & Erne (1966) gave single oral doses
of  100,  200, or  300 mg 2,4-D  amine/kg body weight  to chickens, all
clinical  and  gross  pathological  findings  were  negative,  with the
exception  of a single bird  showing gastritis after the  highest dose.
2,4-D amine was given orally at 300 mg/kg body weight to a second group
of  chickens each day.   One bird died  after 5 days  and was shown  on
autopsy  to have developed  renal and visceral  gout.  The other  birds
were  killed on days 12 or 24 of dosing.  Slight kidney enlargement was
seen,  and there was  an enhancement of  the rate of  2,4-D elimination
with time.

    Bjorn  & Northen (1948) orally dosed white-rock chicks on alternate
days for a period of 4 weeks (12 doses in total) with  an  alkanolamine
salt  formulation of 2,4-D.  All  chicks weighed approximately 50  g at
the  beginning of dosing and doses were adjusted for weight gain of the
chicks through the dosing period.  No effect on weight gain  was  noted
at  doses  of  2,4-D up to 280 mg acid equivalent/kg body weight.  In a
further  study,  single  oral doses of up to 380 mg/kg body weight were
without  effect, but a single oral dose of 765 mg/kg body weight killed
all the birds.

    Whitehead  & Pettigrew (1972a) dosed 28-week-old laying hens daily,
by gelatin capsule, with the butoxyethyl ester of 2,4-D at  either  6.2
or  18.7 mg acid equivalent/bird  for 20 weeks.  There  were no adverse
effects  on egg production,  egg or yolk  weight, egg shell  thickness,
hatchability, or growth rate of the progeny.

    Chickens  were given oral doses of 2,4-D amine salt at 100, 250, or
500  mg/kg body weight  or PGBE ester  at 50, 100,  or 250  mg/kg  body
weight for 10 days in a study by Palmer & Radeleff (1969).  Birds given
2,4-D amine salt did not differ from controls in weight gain,  even  at
the highest dose.  There was similarly no effect from the  lowest  dose
of  the ester.   However, a  growth rate  reduction was  seen with  the
medium  dose  of  the ester (only 19% weight gain relative to a control
weight  gain  of  41%), and  at  the  highest dose,  there was complete
mortality  within  4  days, associated with a weight loss of 13%.  In a
comparable   study,   Palmer   (1972)  dosed   chickens   with    2,4-D
dimethylamine  salt at 25 to  500 mg/kg body weight  for 10 consecutive
days.  There were effects on weight gain at doses of 100 mg/kg or more.
At 100 mg/kg, the weight gain was 38%, compared to a control  value  of
57%.  At 175, 250, and 375 mg/kg, the weight gain was 30%,  similar  to
the  control  value.  At  the highest dose,  three out of  five treated
birds died, and the survivors showed a weight gain of 26%.   There  was
no effect of 2,4-D ethylhexyl ester at 100 mg/kg on weight gain, but at
250  and 500 mg/kg, weight gain was 42% and 36%, respectively, compared
to a control value of 59%.

    Solomon  et al. (1973)  studied the effects  of 2,4-D acid  and two
unspecified  amine salt formulations of 2,4-D.  Pheasants were dosed at
weekly intervals, for 17 weeks, with gelatin capsules containing one of
the preparations at either 75 or 150 mg/bird.  No effects were observed
on  fertility  and  there was  no  increase  in the  number of abnormal
embryos.


Table 11.  Toxicity of 2,4-D to birds
---------------------------------------------------------------------------------------------------------
Species               Sexa  Age         Route  Formulation       Parameter   Concentrationb Reference
                                                                              (mg/litre)
---------------------------------------------------------------------------------------------------------
Mallard duck           M    4 months    oral   acid (technical)  acute LD50     > 2000      Hudson et 
 (Anas platyrhynchos)   M    3-5 months  oral   sodium salt       acute LD50     > 2050      al. (1984)
                       M    7 months    oral   amine salt        acute LD50     < 2000      Hudson et 
                       F    3-5 months  oral   acid (technical)  acute LD50     > 1000      al. (1984)
                            23 days     diet   butoxyethanol     5-day LC50     > 5000 c    Hill et al.
                            17 days     diet   dimethylamine     5-day LC50     > 5000 c    (1975)
                            young       diet   acetamide         100-day LC50   >  500      DeWitt et 
                            adult       diet   acetamide         100-day LC50   > 2500      al. (1963)
                            young       diet   dimethylamine     100-day LC50    2500       DeWitt et 
                            young       diet   butoxyethanol     100-day LC50    5000       al. (1963)
                            adult       diet   butoxyethanol     100-day LC50   > 5000      DeWitt et 
                                                                                            al. (1963)
Japanese quail         M    2 months    oral   acid (technical)  acute LD50     668         Hudson et 
                                                                              (530-842)     al. (1984)
 (Coturnix coturnix          14 days     diet   acetamide         5-day LC50     > 5000 c    Hill et 
        (japonica)           12 days     diet   butoxyethanol     5-day LC50     > 5000 c    al. (1975)
                            20 days     diet   dimethylamine     5-day LC50     > 5000 c    Hill et 
                                                                                            al. (1975)
Bobwhite quail              23 days     diet   butoxyethanol     5-day LC50     > 5000 c    Hill et 
 (Colinus virginianus)       23 days     diet   dimethylamine     5-day LC50     > 5000 c    al. (1975)
                            young       diet   acetamide         10-day LC50    2500        DeWitt et 
                            adult       diet   acetamide         100-day LC50   > 2500      al. (1963)
                            young       diet   dimethylamine     10-day LC50    5000        DeWitt et 
                            young       diet   butoxyethanol     100-day LC50   5000        al. (1963)
                            adult       diet   butoxyethanol     100-day LC50   5000        DeWitt et 
                                                                                            al. (1963)
Pheasant               F    3-4 months  oral   acid (technical)  acute LD50     472         Hudson et 
                                                                              (340-654)     al. (1984)
 (Phasianus colchicus)       10 days     diet   butoxyethanol     5-day LC50     > 5000      Hill et 
                            10 days     diet   dimethylamine     5-day LC50     > 5000 c    al. (1975)
                            young       diet   acetamide         10-day LC50    1000        DeWitt et 
                            adult       diet   acetamide         100-day LC50   > 2500      al. (1963)
                            young       diet   dimethylamine     100-day LC50   5000        DeWitt et 
                            adult       diet   dimethylamine     100-day LC50   > 5000      al. (1963)
                            young       diet   butoxyethanol     100-day LC50   5000        DeWitt et 
                                                                                            al. (1963)
                            10 days     diet   butoxyethanol     5-day LC17     5000        Hill et al. 
                                                                                            (1975)
---------------------------------------------------------------------------------------------------------

    Table 11.  (contd.)
---------------------------------------------------------------------------------------------------------
Species               Sexa  Age         Route  Formulation       Parameter   Concentrationb Reference
                                                                              (mg/litre)
---------------------------------------------------------------------------------------------------------
Chukar partridge      M,F   4 months    oral   acid (technical)  acute LD50     200-400     Hudson et 
 (Alectoris chukar)                                                                          al. (1984)

Rock dove             M,F               oral   acid (technical)  acute LD50     668         Hudson et 
 (Columba livia)                                                               (530-842)     al. (1984)

Chicken               M,F   21 days     oral   free acid         14-day LD50    541         Rowe & Hymas 
                                                                              (358-817)     (1954)
                      M,F   21 days     oral   isopropyl         14-day LD50    1420        Rowe & Hymas 
                                                                             (1127-1789)    (1954)
                      M,F   21 days     oral   mixed butyl       14-day LD50    2000        Rowe & Hymas 
                                               esters                        (1350-2960)    (1954)
---------------------------------------------------------------------------------------------------------
a  M = male; F = female.
b  Acute oral doses are given as mg/kg body weight; all other doses are as mg/kg diet.
c  Dose level of 5000 mg/kg diet produced no mortality.
    Whitehead & Pettigrew (1972b) fed day-old chicken chicks  with  the
butoxyethanol  ester of 2,4-D at  concentrations up to 7500  mg/kg diet
for  3  weeks.   Dietary levels up to 1000 mg/kg had no adverse effect,
but  at 2000 mg/kg  diet 2,4-D ester  reduced the food  consumption and
growth  rate  of the  chicks.  Although there  was no mortality  at the
higher doses, necropsy of birds sacrificed at the end of the experiment
showed  swollen kidneys in all  birds and some mottling  of the spleen.
Bjorklund  &  Erne  (1966) fed chickens with the amine salt of 2,4-D at
500  mg/kg diet.  One bird  died of renal gout  after 5 months  dosing;
autopsy showed hypoplasia (possibly congenital) of the right kidney and
hyperplasia  of  the  left kidney.  Other birds were killed at 1, 2, 9,
or 18 months after dosing began, but there was no consistent pattern to
autopsy findings.

    Erne  &  Bjorklund  (1970)  examined  the  long-term   effects   of
phenoxyherbicides on chickens.  Groups of day-old broiler  chicks  were
given  2,4-D in the drinking water at 1000 mg/litre for up to 7 months.
During the dosing period, chickens were sacrificed at regular intervals
for autopsy and samples were prepared for electron  microscopy.   There
was  decreased  food  and  water  intake  in  dosed  birds.   The  most
pronounced  effect  was  on the  kidney;  there  was noticeable  kidney
enlargement   after  14 days  of dosing and  this increased with  time.
2,4-D  concentrations in body tissues  reached a plateau after  7 days,
with  the highest residue in kidney tissue.  Histologically, the kidney
enlargement  was shown  to be  due to  hypertrophy of  proximal  tubule
epithelium.   These hypertrophied cells, under the electron microscope,
were   shown  to  display   an  increased  mitochondrial   content  and
pronounced  mitochondrial  pleomorphy.   The number  of microbodies was
also increased and nuclear bodies were observed.  These  findings  were
stated  to  reflect  alterations  in  intermediate  metabolism   in the
tubular  cells.  In an earlier study, using chicks dosed similarly with
1000  mg/litre of drinking  water (Bjorklund &  Erne, 1966), the  birds
were  followed through to sexual maturity.  No significant effects were
observed  on  weight  gain, age  at  sexual  maturity, or  onset of egg
production,  but the number of eggs laid was reduced during the first 2
months of egg laying.  The number of birds dying during the  course  of
the  study did not differ from the control value.  Surviving birds were
killed  and autopsied at intervals of between 2 and 18 months after the
onset  of egg laying.  The primary effect was consistent enlargement of
the kidneys.

7.2.3.  Special studies on birds

    Lundholm & Mathson (1983) studied the effect of 2,4-D on  the  ATP-
dependent  Ca2+ binding    of  the  particulate  fraction of  egg shell
gland   mucosa cells  from egg-laying  hens.  This  parameter had  been
found to be a sensitive indicator of potential  shell-thinning  effects
of  chemicals.   They  calculated  a  5-min  IC50,    for  Ca   binding
inhibition,   of   30.7 x 10-8    mmol 2,4-D/litre  incubation  medium.
This   makes   2,4-D  13.5  times less effective  in this respect  than
1,1'-(2,2-dichlorethenylidine)-bis[4-chlorobenzene]   ( p-p' -DDE),   the
major agent causing eggshell-thinning in birds.
    
Percutaneous  absorption of 2,4-D  through the feet  of  red-winged
blackbirds  was measured by Rogers  et al. (1974).  A  24-h exposure to
14C-labelled     2,4-D  at  0.01   mmol/litre  resulted  in   a   blood
concentration of 1.24 x 10-3 mmol 2,4-D/litre.

7.3.  Toxicity to Non-laboratory Mammals

 Appraisal

     Based  on the available data,  no generalization can be  made about
 the  hazard  of  2,4-D to mammals in the field.  Data on voles indicate
 that the herbicide poses no hazard.

    Cholakis   et  al. (1982)  obtained acute oral  LD50 estimates  for
two  species  of  voles by  determining  mortality  14 days  after  the
administration  of  a  single dose  of 2,4-D  acid.  Values  were  2110
(1800 - 2570)   and  2100  (1900 - 2390)  mg/kg  body weight  for males
and   females,   respectively,   of   the   prairie   vole     (Microtus
 orchrogaster).   Values for the  grey-tailed vole  (Microtus canicaudus)
were   1200 (955 - 1150) for  males and 1310  (1010 - 1790) mg/kg  body
weight for females.

    Skokova   (1975)  orally  dosed  24   male  bank voles with  400 to
405 mg/kg body weight (10% of the LD50)   daily for 10 or 20  days  and
examined   reproductive  parameters.   Testis   weight,  an  index   of
spermatogenesis,  and divisions in spermatogonia were all significantly
reduced relative to control values.  Gile (1983) applied a foliar spray
of butyl ester of 2,4-D to a simulated ryegrass ecosystem at  1  kg/ha.
The system included voles, which showed a weight loss after exposure to
2,4-D  when compared to similar  animals in an untreated  system.  This
loss was considered to be the result of protein deficiency.

8.  ECOLOGICAL EFFECTS FROM FIELD APPLICATION

 Appraisal

     No  direct toxic effects, acute or long-term, of 2,4-D applications
 under field conditions on any animals species have been  observed  thus
 far.

     There are, inevitably, indirect effects resulting from the intended
 selective herbicidal properties of the compound.  These  effects  would
 result  from  the use  of any herbicide  or from other  methods of land
 management.   There will, therefore, be effects for mammals, birds, and
 insects   because  of  food   deprivation,  modification  of   habitat,
 requirements for nesting, shelter, etc.

     The  application  of  2,4-D appears  to  present  no hazard  to the
 beneficial epigeal arthropod community.  Physical cultivation present a
 greater  hazard to  sensitive soil  arthropods than  the use  of  2,4-D
 herbicides.

    Oka  & Pimental (1976) observed  increased numbers of insect  pests
and increased occurrence of blight infection in maize  (Zea mays)  crops
treated  with 2,4-D as  the triethanolamine salt.   The crops had  been
treated  with 2,4-D at  0.14, 0.55, or  4.4 kg/ha; 0.55  kg/ha  is  the
normal  rate  of  application for  this  crop.   The number  of  aphids
increased  from 1420  on control  untreated plants  to 2449  on  plants
treated at 0.14 kg/ha, 3116 on plants treated at 0.55 kg/ha,  and  2023
on plants treated at 4.4 kg/ha.  The percentage of plants  attacked  by
the  European  corn borer  (Ostrinia nubialis)  increased  from  63%  on
controls  to  83%  and 70%  for  treatments  at 0.14  and  0.55  kg/ha,
respectively.   Controlled studies also showed an increase in infection
with   fungal  blight.   Laboratory  investigation   of  these  effects
confirmed  that the treated  maize had higher  protein levels than  the
untreated.  This was thought to be the reason for the increased success
of the pests.

    Everts et al. (1986) investigated the effects of various pesticides
on  soil arthropods.  Spiders were found to be a sensitive indicator of
effect.   No side-effects of  the use of  2,4-D amine were  observed on
these  organisms.  Lahr et al.  (1987) showed that spider  numbers were
reduced by ploughing but not by the use of 2,4-D herbicides.

    Matida et al. (1975) examined aquatic organisms in a stream running
through  a mountainous area of 9.4 ha, in Shizuoka prefecture in Japan,
which  had been aerially sprayed with a mixture of 2,4-D and 2,4,5-T at
a  rate of 150  kg/ha.  There was  no effect on  the number or  species
diversity  of  aquatic invertebrates.   Caged  cherry salmon  and  dace
fingerlings  showed no mortality,  abnormal behaviour, or  pathological
change  after spraying.  An extensive  ecological survey of an  area in
Florida  treated with military mixtures of herbicides, including 2,4-D,
revealed  no major change in  species diversity or population  size for
aquatic  invertebrates, fish, a  lizard, or the  beach mouse (Young  et
al., 1975).

    The weevil  Rhinocyllus conicus is used in the biological control of
musk   thistle,  an  invasive  weed.    In  an  investigation  of   the
practicality of combining biological with chemical control, Lee & Evans
(1980)  investigated the toxicity to the weevil of 2,4-D.  They sprayed
musk thistle with 2,4-D at a rate of 4.48 kg/ha.  One week  later,  the
terminal  seed  heads of  the thistle were  covered with cloth  bags to
contain  the  weevils.  The  number of dead  larvae, pupae, and  adults
were counted and compared to the number on unsprayed,  control  thistle
heads.  No significant differences were observed.

    Dwernychuk & Boag (1973) studied the effect of  herbicide  spraying
on  several species  of nesting  ducks (lesser  scaup, gadwall,  white-
winged  scoter, mallard, pintail, and  American wigeon) in Canada.   An
ester   of   2,4-D  was  applied  to  two  islands.   This  application
significantly  reduced the areas  dominated by broad-leaved  plants and
permitted  invasion of these areas by grasses.  Ducks preferred to nest
amongst  broad-leaved vegetation and  avoided grass.  As  the areas  of
broad-leaved  plants disappeared, there was an increase in nest density
in  those broad-leaved areas still  present.  Total numbers of  nesting
ducks  declined  over  the  3-year  study  period.   This  decline  was
attributed,  by the authors, entirely to the effect of the herbicide on
vegetation type.

    Keith  et al.  (1959) studied  the effects  on populations  of  the
pocket  gopher  (Thomomys  talpoides)   of spraying  weedy  rangeland in
Colorado  with  2,4-D, as  the butyl ester,  at 3.4 kg/ha.   Numbers of
gophers  were estimated using two different methods, either by trapping
or  by counting numbers of newly excavated mounds.  Both methods showed
a highly significant difference between sprayed and  non-sprayed  areas
1 year after spraying.  The total numbers of gophers trapped in the two
areas  before 2,4-D application were 101 and 110, respectively.  In the
same  two  areas, 1  year after spraying,  numbers were 117  (untreated
area) and 15 (treated area), respectively.  This represents a  fall  in
gopher  numbers  of 87%  in the treated  area and a  slight increase in
numbers in the unsprayed area.  Newly excavated mounds in  the  treated
area  were only  28% as  numerous as  in control  areas.  Spraying  had
reduced  production  of forbs  (broad-leaved  plants) from   445  kg/ha
before  spraying  to 75  kg/ha afterwards, a  reduction of 83%.   Grass
production had increased by 37%.  The overall reduction  in  vegetation
was 232 kg/ha or 35% on sprayed plots.

    In  a  similar study  by Tietjen et  al. (1967), 2,4-D  butyl ester
applied  at 3.4 kg/ha  to identical high-altitude  rangeland  initially
reduced  forb density and  gopher populations.  Pocket  gopher  numbers
were reduced by between 80% and 90%.  Both forbs and gopher populations
remained  low in one treated area but not in a second one.  The decline
in  gopher numbers was considered by the authors to be a result of food
deficiency; the grasses available represented a marginal diet  for  the
animals.  This decline was not due either to movement of animals out of
the area or to the direct toxic effects of the herbicide.  Reduction in
numbers was, therefore, primarily a result of reduced breeding success.

    Johnson & Hansen (1969) studied the after-effects on  wild  mammals
of  treating perennial forb  and shrub/grass ranges  with 2,4-D  either
aerially  or from a  ground rig at  rates of 2.2  or 3.4 kg/ha  using a
diesel-oil  carrier.   The density  and litter size  of the deer  mouse
(Peromyscus  maniculatus) was little affected by the treatment, but the
densities  of  northern  pocket gophers  (Thomomys talpoides)  and least
chipmunks (Eutamias  minimus) were  reduced.  Montane  voles   (Microtus
 montanus)  increased their abundance in  treated perennial forb  range.
Gopher   and  vole  populations  returned   to  normal  with  the   re-
establishment  of forb dominance.   Density changes were  considered by
the  authors to be primarily due to changes in availability of food for
the  gophers, availability of  both food and  cover for chipmunks,  and
availability of a close-canopied grass cover for the voles.  There were
no direct toxic effects of the herbicide.

    Fagerstone  et  al. (1977)  observed  two colonies  of black-tailed
prairie dogs  (Cynomys ludovicianus)  in North America and the effect of
spraying  their feeding areas  with 2,4-D.  The  herbicide was  applied
initially  as the dimethylamine salt, followed by two further sprays of
the butyl ester after 1 month and 1 year.  All  applications  of  2,4-D
were  at 2.2 kg/ha.  One colony lived in a sprayed area, initially rich
in broad-leaved plants.  The second colony lived in an  unsprayed  area
poor  in  dicotyledonous  plants and  rich  in  grass.  The  effect  of
spraying  was,  therefore,  to make  the  treated  area similar  to the
control area with less broad-leaved herbage and less cover.   Prior  to
treatment,  the first colony preferentially ate the forbs; diet was 73%
forbs and 5% grass.  After spraying, they ate 9% forbs and  82%  grass.
The  control  colony  ate a  similar  diet  to the  first  colony post-
treatment,  i.e., mostly grass.  Prairie dogs remained in the same area
after treatment with herbicide and there was no evidence of starvation.
Body  weight  was  maintained, activity  was  comparable  to  the  pre-
treatment level, and reproduction was unaffected.

    Spencer  &  Barrett (1980)  monitored  the population  response  of
meadow voles  (Microtus pennsylvanicus)  to application of  2,4-D as the
N-oleyl    1,3 propylenediamine salt at  567.5 g/ha.  Two 0.4-ha  plots
were  compared, one sprayed  and the other  untreated.  The  population
fluctuation  of the  voles was  monitored for  6 months,  from June  to
December,  a period  spanning the  breeding season.   The control  area
reached  a  population  peak of 116 animals on 6 November; a peak of 68
voles was reached on 9 October in the treated area.  There was a skewed
sex  ratio in the  treated area, mainly  because of a  reduced survival
rate  in females.  Voles  in the treated  plot were  protein-deficient,
compared to controls

9.  EVALUATION

    In  evaluating the environmental  hazards of 2,4-D,  the  following
general points should be borne in mind:

    (a) the   chlorinated  dibenzo- p -dioxins    (CDDs) are  present in
    2,4-D  only in trace  amounts which are  difficult to separate  and
    identify;

    (b) 2,4-D is rapidly degraded in the environment;

    (c) the   environmental   effects   are  indirect,   and  are  the
    consequence  of vegetation diversity being modified; care should be
    taken  to  avoid  unintentional  vegetation  damage;  the  mode  of
    application  and  the  formulation should  be  carefully  selected;
    esters  should  be  avoided  in  aquatic  applications  (because of
    toxicity to aquatic organisms);

    (d) there  are  limited  data on  the  effects  of 2,4-D  and  its
    formulations  on  communities  of organisms;  hazard assessment is,
    therefore, often by extrapolation from single species studies;

    (e) minor   adverse  effects  shown   in  laboratory  studies   on
    terrestrial organisms have resulted from exposures far in excess of
    likely exposures in the field.

9.1.  Aquatic Organisms

    Sources of exposure of aquatic ecosystems to 2,4-D  include  direct
application,  run-off, and spray drift.  Because of low adsorption rate
and rapid degradation, the herbicide is not accumulated in compartments
of the aquatic system.

    2,4-D acid and its salts are less toxic to aquatic  organisms  than
are the esters.

    2,4-D acid and its salts are of low to moderate toxicity to aquatic
organisms.   However,  the  growth  and  nitrogen  fixation   of   some
cyanobacteria   (blue-green   algae)   are  inhibited,   but   only  at
concentrations  above levels expected  from direct application  of  the
herbicide  to  water.   These microorganisms  are  the  source of  most
nitrogen  in wet tropical  soils.  This inhibitory  effect could be  of
concern  when these  compounds are  applied to  rice fields  at a  high
dosage.

    Because  of  the toxicity  of  esters, particularly  the  propylene
glycol  butyl  ether  ester, for  early  life-stages  of  several  fish
species, they should be regarded as hazardous to aquatic ecosystems.

9.2.  Terrestrial Organisms

    2,4-D  does  not  persist in  soil  and  other compartments  of the
terrestrial environment.

    Nitrogen-fixing  microorganisms appear to be particularly sensitive
to 2,4-D; this might be especially important in tropical soils.

    Some terrestrial invertebrates have shown adverse effects, but only
at high exposure levels.  Therefore, 2,4-D does not constitute a hazard
to this group of organisms.

    2,4-D  has  low  acute  toxicity  to  birds,  as indicated  by  the
LD50.   Most studies on birds  and  their eggs have  been  conducted at
exposures  exceeding those that  could be expected  in the field;  even
under  these  conditions,  no  significant  adverse  effects  have been
observed.

    Under field conditions, 2,4-D does not cause direct  toxic  effects
on animals.  However, the change of species composition  and  structure
of the vegetation, resulting from the use of this herbicide,  leads  to
indirect effects on terrestrial ecosystems.  This indirect effect would
also result from the use of any herbicide or from other methods of land
management in either temperate or tropical regions.

                *                *               *

    There is evidence only for minor effects on the environment arising
from  the use of 2,4-D, as long as the following simple recommendations
are followed:

    (a)  amine formulations,  rather than  esters, should  be  used  to
    control aquatic weeds;

    (b) accidental spread of the herbicide to other  vegetation  should
    be avoided;

    (c)  the margins of agricultural land should be left untreated with
    herbicide  to avoid even  the indirect effects  of the material  on
    wildlife.

10.  RECOMMENDATIONS FOR FURTHER RESEARCH


    There  are  indications that  2,4-D  affects nitrogen  fixation  by
algae.  Since this is the major source of nitrogen in  tropical  soils,
it   is  recommended  that   this  should  be   further   investigated,
particularly with reference to varying soil conditions.  This should be
extended to a study on the functioning of a rice-paddy at the ecosystem
level.

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of  Puntius ticto  (Ham.).  J. environ. Biol., 5: 249-254.

WATSON, J.R.  (1977)  Seasonal variation in the biodegradation of 2,4-D
in river water.  Water Res., 11: 153-157.

WELP,  G.  &  BRUMMER, G.   (1985)   [The  Fe(III)-reduction test  -  a
simple procedure to determine the effects of environmental chemicals on
the   microbial  activity  in  soils.]  Z.   Pflanzenernaehr.  Bodenkd.,
148: 10-23 (in German).

WHITEHEAD,  C.C.  & PETTIGREW,  R.J.   (1972a)  The  subacute  toxicity
of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid
to chicks.  Toxicol. appl. Pharmacol., 21: 348-354.

WHITEHEAD,  C.C.  &  PETTIGREW,  R.J.   (1972b)   The  effect  of  2,4-
dichlorophenoxyacetic  acid on laying  hens.  Br. poult. Sci., 13:  191-
195.

WHITNEY,     E.W.,     MONTGOMERY,     A.B.,    MARTIN,     E.C.,     &
GANGSTAD,  E.O.  (1973)   The effects  of a  2,4-D application  on  the
biota and water quality in Currituck Sound, North  Carolina.    Hyacinth
 Control J., 11: 13-17.

WHO   (1984)    Environmental  Health Criteria  29: 2,4-dichlorophenoxy-
 acetic acid (2,4-D), Geneva, World Health Organization, 151 pp.

WOODWARD,   D.F.   (1982)   Acute   toxicity  of  mixtures   of   range
management  herbicides to cutthroat  trout.  J. Range Manage., 35:  539-
540.

YOUNG,  A.L.,  THALKEN,  C.E., &  WARD,  W.E.   (1975)  Studies  of  the
 ecological  impact of repetitive  aerial applications of  herbicides on
 the ecosystem of Test Area C-52A, Elgin AFB, Florida. Florida Air Force
Armament  Laboratory, Elgin Air Force Base, 126 pp. (Report No.  AFATL-
TR-75-142).



    See Also:
       Toxicological Abbreviations