Development of short-term
exposure tests for marine
macroalgae for use in effluent
testing

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FINAL REPORT FOR COOPERATIVE AGREEMENT f CR812070-01

DEVELOPMENT OF SHORT-TERM EXPOSURE TESTS FOR MARINE

       MACROALGAE FOR USE IN EFFLUENT TESTING
                         by
                  Glen B. Thursby
                 Botany Department
             University of Rhode Island
               Kingston, R.I.  02881
                  Project Officer

                  Richard Latimer

         Environmental Research Laboratory
        U.S. Environmental Protection Agency
             Narragansett, R.I.  02882
                 Noverraber 26, 1986

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                               DISCLAIMER



This document has not been peer and administratively  reviewed within EPA




and is for internal Agency use/distribution only.
                                  ii

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                                OVERVIEW


     A relatively fast,  simple and inexpensive  toxicity test method has


been  developed for the marine red macroalga  Champia  parvula,  to assess


chronic effects of pollutants to marine macroalgae.   The method has been


used  to generate toxicity data for water quality  criteria.   The  test


method  has  previously  been evaluated with  heavy metals  and  cyanide,


arsenate and arsenite, as well as ten different  organic compounds.  This


test method showed that sexual reproduction is the best endpoint to  use


for Champia.   If sexual reproduction is used as the  sole endpoint, then


plants  should only have to be exposed to toxicants for a few days (long


enough  to show any effects on fertilization).   The  previous  toxicity


test  with Champia lasted two weeks,  however,   a modification has  been


developed  in which females and males are exposed together to a toxicant


for only two days.  The procedure has been used  successfully with single

                   »
compounds and a variety of complex effluents.


     Initial  steps  were  also taken in the  development  of  a  similar


short-term test with the brown alga,  Laminaria  saceharina.   Laminaria


represents another phylum  of algae than Champia, and has the additional


advantage of being both economically and ecologically important.  Sexual


reproduction  in  Laminaria  has already been shown to be  sensitive  to


petroleum  products.   For the current study, the feasibility of  using


Laminaria  as a routine toxicity test species was verified.   The  large


adult  sporophyte of Laminaria is difficult to maintain in  the  labora-


tory.  However, the male and female gametophytes are  microscopic and are


easily cultured.


      The   Office  of  Water's Permits Division of  the  Environmental


Protection  Agency  needs  toxicity  test  procedures for  marine   and


                                    iii

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eatuarine  species.   The  procedures  are needed   to  characterize  and




quantify  the  toxicity  of effluent discharges  by National  Pollutant




Discharge  Elimination System permittees.   The  tests must yield chronic




data in a relatively short time period (7  days or  less).   The data must




be both scientifically sound and legally defensible.   At the same time,




it  is  necessary  for  the methods to  be simple and  cost  efficient




(requiring  standard  hardware  and laboratory   facilities).   The  test



species should be readily available, and testing should be practical for




both on-site and off-site.  All of these requirments are met by the test




methods developed for the marine algae, Champia parvula and   Laminaria



saccharina.




     This  report  covers  work completed  during the  time  period  from




November 1,  1984 through October 31,  1986.  The  specific objectives of




the cooperative agreement were:




  I.  Champia parvula



     A. Write  final guidance manual for conducting short-term  chronic




        tests.



     B. Compare  data  from  above  test procedure with  that  from  the




        existing two-week test.



     C. Test  short-term  test  in field  and  laboratory  with  complex




        effluents.



  II. Laminaria saccharina



     A. Test  feasibility of Laminaria saccharina  as a routine  toxicity



        test species for short-term tests.




     B. Compare the sensitivity of Laminaria with  that of Champia.
                                   iv

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     The  techniques  developed  during this cooperative agreement were



presented at three effluent monitoring workshops.   The Champia technique



was presented at EPA1s Narraganaett, Rhode Island  laboratory in October,



1985,  and at EPA'3 Gulf Breeze,  Florida laboratory in February,   1986.



The  Laminaria technique was presented at the workshop held in  Newport,



Oregon, in October, 1986.

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                               TABLE OF CONTENTS
PART I.  GUIDANCE MANUAL FOR CONDUCTING SEXUAL REPRODUC-




   TION  TESTS WITH THE MARINE MACROALGA CHAMPIA PARVULA




   FOR USE IN TESTING COMPLEX EFFLUENTS	
PART  II.   COMPARISON  OF  SHORT- AND LONG  TERM  SEXUAL




   REPRODUCTION  TESTS WITH THE MARINE RED  ALGA  CHAMPIA




   PARUVLA	
   THAT OF CHAMPIA
                                                                  PAGE
                                                                   1-1
                                                                   II-l
PART III. SUMMARY OF RESULTS FROM TESTING COMPLEX  EFFLUENTS .....






PART  IV.  PRELIMINARY  GUIDANCE  MANUAL  FOR  CONDUCTING




   SEXUAL  REPRODUCTION TESTS WITH THE MARINE  MACROALGA,




   LAMINARIA  SACCHARINA,  FOR  USE  IN  TESTING  COMPLEX




   EFFLUENTS ............. . ......................................    IV_1






PART  V.  COMPARISON OF THE SENSITIVITY OF LAMINARIA  WITH
                                   vi

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                           PART I
GUIDANCE MANUAL FOR CONDUCTING SEXUAL REPRODUCTION TEST WITH




            THE MARINE MACROALGA CHAMPIA PARVULA




            FOR USE IN TESTING COMPLEX EFFLUENTS
                             1-1

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TABLE OF CONTENTS




                                                                page




INTRODUCTION	  3




BACKGROUND	  5




MAINTENANCE OF STOCK CULTURES	  8




ARTIFICIAL SEAWATER	 12




SALINITY ADJUSTMENTS	 14




PREPARATION OF PLANTS FOR A TEST	 15




TEST CHAMBERS	 18




TEST CONDITIONS	 19




PROTOCOL	 22




STATISTICAL TREATMENT OF DATA	 25




CRITERIA FOR ACCEPTABILITY	 26




REFERENCES	 27




ADDENDA	 29

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                              INTRODUCTION




     Seaweeds have been considered  less useful for toxicity testing than




microalgae  (Jensen,  1984),  and   microalgae are often considered  less




sensitive  than aquatic animals (Kenaga  and  Moolenaar,  1976;  Kenaga,




1982).   Therefore,  one  could easily come to the erroneous  conclusion




that  toxicity  testing with seaweeds is not necessary.  As recently  as




1983 the statement was made that "seaweeds seem to be rather insensitive




to  many chemicals and will probably survive pollution better than  many



other organisms in the marine environment" (Jensen, 1984).




     A two-week toxicity test method has already been developed for  the




maeroalga,  Champia parvula, to assess chronic effects of pollutants  to




marine  seaweeds (Steele and Thursby,  1983).   The test has  previously




been  evaluated with heavy metals (Steele and Thursby,  1983),  arsenite




and  arsenate  (Thursby and Steele,  1984),  and ten  different  organic




compounds (Thursby,  et al.,  1985).  This test method shows that sexual




reproduction  is generally the most sensitive and practical endpoint  to




use for Champia.



     Pollution  assessments with macroalgae must take reproduction  into




consideration  if  an accurate picture of the potential harm  is  to  be




drawn.  Previous conclusions about  seaweed sensitivity were based large-



ly on growth as the endpoint.  The  ability to measure the sensitivity of




seaweeds  to toxicants increases when sexual reproduction is used as  an



endpoint;  and  can be greater than many aquatic animals that have  been



tested.   This has been shown for Champia parvula (see above references)



as  well  as  for  the brown macroalgae Fucus  edentatus  and  Laminar!a




saccharina (Steele and Hanisak, 1978).



     The  above toxicity test with  Champia parvula is a two-week  growth



and  reproduction study and requires that the cultures remain  unialgal.






                                  1-3

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This  time period makes the test unaccepatable for testing  complex  ef-




fluents  and  their receiving waters.   Fine-filtering,  which would  be




necessary to remove unwanted microalgae,  could change the character  of




the  effluent  or receiving water.   Any microalgae introduced with  the




effluent  would compete with Champia for light and nutrients,  thus  in-




fluencing  Champia's growth rate.   However,  if sexual reproduction  is




used as the sole endpoint, then plants only need to be exposed for a few



days  (long  enough to show effects on fertilization).   Any  effect  of




other organisms on the growth rate of Champia would not be serious since




interest would only be in whether sexual reproduction had taken place.




     Sexual  reproduction  was  selected as the  endpoint  for  effluent.




testing for several reasons.  It had previously proven to be a sensitive




endpoint  from  the two-week toxidty test procedure using  single  com-




pounds.  .A sexual reproduction test for toxicity could be short  enough




to  fit  the  tine-constraints for tests used in the  effluent  program.




Finally,  Champia is an annual plant and inhibition or absence of sexual




reproduction  reduces or eliminates the next stage in its life  history.




Total  absence of cystocarp formation is the easiest endpoint to  inter-




pret  as far as field populations are concerned.   In most of   the  red



algae  each fertilization results in the formation of a new life history




stage,  the carposporophyte, "parasitic" on the female and housed within



the cystocarp.  Each carposporophyte is capable of producing many spores



(perhaps a hundred or more in the case of Champia).  This characteristic



makes it difficult to interpret the biological significance of a statis-



tical  decrease  in  the number of cystocarps or  an  arbitrary  percent



decrease such as 507..  Absence of reproduction leaves no doubt about its




biological significance.
                                  1-4

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                               BACKGROUND
     This paper describes a method which uses sexual reproduction, after
a short-term exposure to effluents,  to estimate chronic  toxicity.   In
brief, the method consists of exposing males and females to effluents or
receiving waters for two days, followed by a 5- to 7-day recovery period
in  control medium.   The recovery period allows time for any cystocarps
to  mature.  At the end of the recovery period the number of  cystocarps
per plant are counted.  The goal for Champia within the effluent program
is to use absence of sexual reproduction as the  endpoint.   Statistical
differences (or other "cut-offs"),  although more difficult to interpret
ecologically,  can also be included to more easily make comparisons with
other  marine  species in the program.   In addition,  the reporting  of
concentrations  that cause the total absence of sexual reproduction  and
statistical differences will give some idea of the steepness of the dose
response curve.
                 r
     The  method described here has been used for both single  compounds
and  complex effluents.   Tests have been conducted on-site in a  mobile
laboratory and at the EPA's Environment1 Research Laboratory,  Narragan-
sett,  HI.  Several different types of effluents have been tested. These
included  one  from a pulp mill;  two industrial  sites  that  discharge
effluents  containing  heavy metals;  five industrial sites  discharging
organically contaminated effluents,  including pesticides and dyes;  and
17  different sewage effluents.   In addition several  receiving  waters
have also been tested.   In all, more than 30 tests have been performed.
When  basing the test endpoint on the absence of reproduction,  the pulp
mill effluent had its effect between 1.0 and 2.5 7. effluent;  the  heavy
metal  effluents  had  a range of effect of 0.054 to  0.50  7.  effluent;
                                 I- 5

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organic  effluents  had a range of 3 to 20 7. effluent (generally <5  7.};




and  the sewage effluents ranged from 2.5 to 25 7. effluent (generally  <




10  7.).   Receiving  water effects have been detected  and  ranged  from




little  or  no  effect on sexual reproduction to  total  elimination  of



sexual reproduction.




     Nine  single compounds have been used to compare effects on  sexual




reproduction using the two-week test and the two-day exposure.   Several




different  cut-off points for the endpoint of sexual  reproduction  from



the  two-day exposure were compared against the  no sexual  reproduction




(HSR) endpoint from the two-week test (Thursby and Steele,  1986).  From




among these comparisons 37.-of-the-control (95% or greater decrease) gave




the  best  estimate of the NSR effect for the two-week test  (Table  1).




Therefore,  cystocarp  counts at < 5% of the control are considered zero




for the short-term exposure test.




     The  sexual reproduction,  two-day exposure test has been developed




as a static,  non-renewal test (although daily media changes are  possi-




ble)  for effluents and receiving waters.   The method is easy and  cost



efficient  to perform.   Stock cultures are maintained in the laboratory




with  standard laboratory equipment,  therefore,  plant material can  be



available  year-round.   The  test procedure is intended to be  used  to



estimate chronic effects of complex effluents on marine oacroalgae.
                                   1-6

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Table  1.   Comparison of the short-term exposure test and the  two-week
test  using single compounds.   Values  listed are the geometric means of
the effect and no-effect concentrations in ug/L.   Effect is defined  as
either  £  57.  of the control (short-term exposure test)  or  no  sexual
reproduction (two-week test).
COMPOUND
Arsenite
Cadmium
Copper
Silver
Benzene
Isophorone
Pentachloro-
SHORT-TERM (STE)
232
>iooa
7.7
0.92
73,600b
107,050
10,170
TWO- WEEK (TWT)
139
77
7.7
1.50
73,600
107,050
10,170
RATIO (STE/TWT)
1.67
>1.30
1.00
0.61
1.00
1.00
1.00
 ethane

Pentachloro-       465                  465                   1.00
 phenol

Toxaphene          .140                  140                   1.00


aSTE was run in polystrene cups, in 125 Erlenmeyer flasks and in 500 mL,
 screw-capped flasks with 400 mL of medium.  The results were always the
 same.   The STE may not work for cadmium because it is a slow  toxicant
 and two days is not enough time to see its full effect.

 STE  was run in 400 mL of medium in 500 mL,  screw-capped flasks.   The
 STE  did  not compare well with the TWT when the  cups  were  used.   A
 larger  volume may be necessary when working with highly volatile  com-
 pounds .
                                  1-7

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               MAINTENANCE OF STOCK CULTURES OF CHAMPIA PARVULA


        Plants of Champia parvula  (C. Agardh) Harvey (Rhodophyta) are bushy


   and  5  to  10 ca  tall in the field.   The main axis  and  branches  are


   cylindrical,  hollow and septate.  Champia's life history is an alterna-


   tion of isomorphic generations  (Fig.  1).   The clone presently used was


   isolated from Rhode Island waters in 1979.  It is probably not essential


   to have a standard clone,  however,  some experience is required to iso-


   late  new clones from the field into unialgal culture.   Unialgal  stock


   cultures are necessary to maintain healthy,  actively growing plants for


   use  in testing.   7egetatively propagated plant material from the  1979


   clone is available from the U.S.  Environmental Protection Agency, South


   Ferry Road, Narragansett, RI  02882.
tetraaporonqia—?
                                            MALE
                                                         spermatic
                                                         a
      TETRASPOROPHYTE
                                      FEMALE
                                     5mm
                                                        fertilization
                                                       ^cystocarp
         Figure  1.   Life  history  of  the marine red alga Champia parvula.
                                     1-8

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     Male  and   female  plants of Champia can be  maintained   easily  in




unialgal  culture in the  laboratory.   New cultures can be  started  from




excised branches,  making it possible  to maintain clonal material indef-




initely.  No special preconditioning is required to induce  reproduction.




Under the conditions listed below,  male gaoetophytes produce  spermatia




continuously and females  are always receptive.  Thus, plant material can




be available at  any time  for testing.




     Laboratory  cultures  of  Ghanpia provide test  plants  of  similar



preconditioning.   Unialgal stock cultures of both males and females are




maintained in separate, aerated 1000 mL Erlenmeyer flasks containing 300




mL  of the culture medium.   The choice of this  flask is one of prefer-




ence rather than necessity.  All culture glassware should be acid-strip-




ped in 10 to 12  7. HC1 and rinsed in deionized water after washing.  This




is necessary since many detergents can leave a residue that is toxic  to




Champia.  The  culture  medium  is made from natural seawater  to  which




additional  nutrients  are added (Table 2).   The  nutrients  used  with



artificial  seawater  can also b« used (see ARTIFICIAL  SEAWATER).   The




seawater  is autoclaved for 30 min at 15 psi.   The culture  flasks  are




capped  with  aluminum foil and autoclaved dry,  for  10  min.   Culture




medium  is  made up by dispensing seawater into the sterile  flasks  and



adding the appropriate nutrients from a sterile stock solution.   Alter-




nately,  liter   flasks could be autoclaved with the seawater already  in



them.   Sterilization  is used to prevent microalgal contamination,   and




not to keep cultures bacteria-free.



     We  recommend  that  several cultures of both males and  females  be



maintained  simultaneously to keep a constant supply of  plant  material



available.   Some  cultures should be at different stages of development




(i.e.,  with  different amounts of  tissue per  flask).   Initial  stock
                                  I-9

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cultures  should be started weekly with about twenty 1.0 cm branch tips.

Cultures are gently aerated through sterile, cotton-plugged, disposable,

polystyrene 1 mL pipets.  Cultures are capped with foam plugs and alumi-

num foil and illuminated from the side with 75 uE m"  s"  of  cool-white

fluorescent light on a 16:3, light:dark cycle.  The temperature is 22 to

24 'C and the salinity 28 to 30 °/,a.  Media are changed once a week.
Table  2.   Recipe for additional nutrients to be added to natural  sea-
water  for stock cultures and test medium.   Both EDTA and trace  metals
have  been  omitted.   The concentrated stock solution is autoclaved  at
standard temperature and pressure for 15 minutes (the pH is adjusted  to
2.0 prior to autoclaving to prevent precipitation).
   COMPOUND                           AMOUNT/LITER
                      TEST MEDIUM CONC.           CONCENTRATED STOCK*
Na»03
N«H2P04«H20
Iron
Vitamins
B12
Biotin
Thiamine*HCl
9.35 mg
0.62 mg .
2.6 ug
0.06 ug
0.06 ug
12.5 ug
3.74 grams
0.25 grams
1.04 mg
10 mL6"
 use 0.25 mL/100mL (2.5 mL/L) for test medium concentrations and 10 mL/L
 for  stock  cultures.   For test medium only,  add 0.25 mL/100mL  of  a
 sodium  bicarbonate  solution.   A  stock solution of 60  mg/mL  sodium
 bicarbonate  is  prepared by autocalving it as a dry  powder  and  then
 dissolving it in sterile deionized water.

 Iron  stock  solution prepared by dissolving 1 g iron powder in 10  mL
 concentrated HC1 and diluting to 1 liter with deionized water.  Accept-
 able  stock  soultions  could  also be  made  with  ferric  or  ferrous
 chloride.

Vitamin  stock  solution  autoclaved separately in 10  mL  sub-samples.
 Each  10  mL contains 24 ug B12,  24 ug biotin and 5  mg  thiamine-HCl.
 Adjust pH to ca 4.0 before autoclaving for 2 min.
                                  1-10

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     About half of the plants should be discarded  (or  placed  into  anoth-




er  culture vessel) with each weekly medium change to  reduce  the   amount




of biomass as the plants grow.  At the end of  three weeks  plants will be




ready  to use for testing.  Readiness is defined as having enough   plant




material  to perform at least one test.   With this procedure,  actively




growing  plants  will be continuously available.    The total  number  of




cultures maintained will depend on the expected frequency  of  testing.




     A stock culture should not be used as a source of test material  if



the  plants appear to be stressed  or undernourished.   Under conditions




of stress the tips of the branches will turn "pink" and the older  tissue




will generally be much paler. In addition, the sterile hairs  of stressed




plants will appear stubby, especially near the branch  tips.   The tricho-




gynes of stressed female plants will also be stunted or absent. This can




be  evident even in plants that do not have any apparent   color  change.




Under conditions of nutrient deficiency (resulting usually from too much




plant  material in the culture flask or too long   since the last   medium




change) the entire plant will turn pale yellow.  If the stress is  severe




enough  the  older tissues (main axes) or occasionally the branch tips




will turn white (evidence of necrbtic tissue).   If cystocarps are pres-



ent  on females in the stock cultures,  the plants  are not suitable  for



testing  (this usually happens as a result of  contamination by males  or




water from male cultures).
                                 I-11

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                          ARTIFICIAL SZAWATER



     Because  salinity  adjustments  will be necessary  in  many   complex



effluent tests, an artificial seawater recipe that yields  good growth of



Champia is desirable.   An artificial seawater would also  make the   test



method more readily available to  laboratories that do not  have access to



clean  natural  seawater.   Some  commercial preparations  are  toxic to



Champia (presumably due to the presence of high concentrations of  trace



metals  that are in the commercial grade of salts used  in  their prepara-



tion).  We have had success with  artificial seawater using GP2 (Spotte,



et  al.,  1984).   Plants grow and reproduce well and have the   correct



morphology  in  GP2,  although they may be slightly smaller in  diameter



than plants grown in natural seawater.  Plants require  approximately two



weeks  to acclimate to the artificial seawater.   The recipe  for  arti-



ficial seawater with GP2 is listed in Table 3.



     A comparison between the sensitivity to toxicants  in  the GP2 medium



with  that in natural seawater has only been made for   copper.    Results
suggest  that plants grown and  tested  in GP2  are  slightly  less  sensitive




to  copper  than plants from natural seawater.    This may  be due   to  an




acclimation to higher  levels of heavy  metals  in the GP2 medium  (from  the




reagent  grade salts).   However,  comparisons with more   compounds   are



needed  before we can  conclude  that plants grown  in GP2 are always  Less




sensitive.



     During  the  months  of June,  July and  August the quality of   the



seawater  that  we normally use to culture and test Champia  parvula  is



often poor.   We have, however, been able to  obtain excellent growth  and



reproduction  from Champia during this period of  time by mixing GP2   and



natural seawater in a  50:50 ratio.   The plants required no  acclimation




period to this mixture.





                                 1-12

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Table 3. Recipe for artificial seawater using GP2. The concentrations of
the  salts have been adjusted to give a final salinity of  30a/«o.   The
original  recipe  calls  for autoclaving anhydrous and   hydrated  salts
separately to avoid precipitation.   However,  if the sodium bicarbonate
is autoclaved separately (dry),  then all of the salts can be autoclaved
together.   Since no nutrients are added until needed,    autoclaving is
not critical for effluent testing.  To minimize microalgal contamination
the  artificial seawater should be autoclaved when used for  stock  cul-
tures.   Autoclaving  should be for at Least 10 min for 1 liter  batches
and  20  min  for 10 to 20 liter volumes (at  standard  temperature  and
pressure).


        COMPOUND                        GRAMS/LITER*
NaCI
Na2S04
KC1
KBr
Na2B40?.10H20
MgCl2-6H20
CaCl2.2H20
SrCl2-6H20
NaHC03b
21.03
3.52
0.61
0.088
0.034
9.50
1.32
0.02
0.17
Generally made in 10 to 20L batches.

 A  stock  solution of 68 mg/mL sodium bicarbonate is prepared by  auto-
 claving it as a dry powder and then dissolving it in sterile  deionized
 water.  For each liter of GP2 use 2.5 oL of this stock solution.
                                 1.13

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                          SALINITY ADJUSTMENTS

     We have used natural seawater brine (made by evaporation to ca. 100

"/oo),  GP2 brine (GP2 made to 3x strength),  and concentrated GP2 salts

added  separately,  to  adjust  brackish receiving waters  to  30  */««•

Salinity  adjustments with dry salts are usually too  cumbersome,  espe-

cially for use in a mobile laboratory.   If the initial salinity of  the

receiving  water is >15 °/a«,  then plants grown in natural seawater can

b« used with any of the above methods of salinity adjustment.   However,

if the initial salinity of the receiving water is <15 '/„<>»  then plants

previously acclimated to artificial seawater are recommended.  In either

case,  a  control  using clean natural seawater diluted  with  deionized

water to the lowest salinity to be tested (then adjusted upwarded to  30

*/ao with one of the techniques above) should be used.   If the salinity

range  of  the  receiving waters to be tested from a given  location  is

great,  then  it  is advisable to make up diluted  controls  at  several
                  »
salinities.

     Plants  grown  in  artificial seawater have always done  well  when

placed  into natural seawater (i.e.,  no acclimation  period  required).

However,  plants  grown  in natural seawater do not always do well  when

placed  into  artificial seawater..   This is true even for the  two  day

exposure period.
                                  I_L4

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                    PREPARATION OF PLANTS FOR A TEST




     Stock  cultures  should be checked  for  their  readiness  for   use   in




toxicity tests.   Females can be checked by  examining  a  few  branch  tips




under  a  compound microscope (100 X or  greater).   Several   trichogynes




(reproductive hairs to which the spermatia attach) should be easily seen




near the apex (Fig. 2).  Male plants should  be visibly producing  sperma-




tia.   This can be checked by placing some male tissue in a  petri dish,




holding  it  against a dark background and looking for the   presence   of



spermatial sori (Fig.  3).   Another way is  to examine the males  under a




compound microscope.  A mature sorus can be  easily identified by  looking




at the edge of the thallus (Fig.  4).   A final,  quick  way  to determine




the relative "health" of the male stock culture is to  place  a portion  of




a  female  plant into some of the water  from the male  culture for a  few




seconds.   Under  a  compound microscope numerous  spermatia should   be




attached to both the sterile hairs and the trichogynes (Fig. 5).




     Once  cultures  are determined to be usable for   toxicity  testing,




branch tips should be cut into their final size.   For females, cut 7  to




10 mm branch tips, enough for 5 per treatment chamber  (try to be  consis-




tent in the degree of branching;  see Fig. 6). The cutting can be easily



done  with fine-point forceps with the plants in a little seawater in   a



petri dish.   Repeat for males, except cut 1.5 to 2 cm branches and only




one per treatment chamber.   The males should visibly  be producing sper-



matia (i.e. two or more spermatial sori present).  Cut the females first




to  minimize the chances of contaminating them with water from the  male




stock cultures.
                                 1-15

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                                    sterile  hairs
                                           -^trichogynes
                     1 mm
Figure  2.    Apical  tip  of female  branch of  Champia  parvula  showing
     sterile  hairs and trichogynes  (reproductive hairs).  Sterile hairs
     aze wider and generally ouch longer than trichogynes.   They  also
     appear  to  be hollow except  at their apex.   Both types  of  hairs
     occur   around the entire circumference  of the thallus but are  seen
     easiest at the "edges".   Receptive trichogynes occur only near the
     branch tips.
                1 cm
                                      spermatial
                                             sorus
Figure 3.   A portion of a male  thallus  of Champia parvula showing sper-
     matial  sori.    The sorus areas  are generally slightly  thicker  in
     diameter and a little lighter  in color.
                                 1-16

-------
                          cuticle
               ICO^m
                                   0  ^
                                ^  0    spermatia
                                      «
                                    thallus surface
Figure 4.   A close-up of a portion of a spermatial  sons, note the rows
     of cells that protrude from the thallus  surface.
                                         spermatic
Figure  5.   Apical  tip  of a female of  Champia  parvula  that  had  been
     "dipped" in water from a mala culture.   The sterile and  reproduc-
     tive  hairs  are  covered with spermatia.    Note  that  older  hairs
     (those  more than about one mm back  from the apex) have few  to  no
     spermatia attached to them.
                                     1 cm
Figure  6.   The size and degree of branching  that  is  generally used for
     the  female  starting plants.    Occasionally the  branches  will  be
     longer near the tip,  try to be consistent  in  the degree of branch-
     ing since the receptive trichogynes  are at  the branch tips.  There-
     fore,  the  more  variation in the degree of  branching,  the  more
     variation in the potential number of cystocarps per plant.
                                1-17

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                             TEST CHAMBERS




     Most  testing to date has been performed with 100 mL in ca.  110 mL




polypropylene cups with fitted polyethylene caps (Falcon*).   These cups




offer the advantage of being disposable,  and their wide opening  allows




easy access to plants for transferring to recovery bottles.   The use of




these cups is out of preference rather than necessity.  Successful tests




have  also  been run in 125 mL Erlenaeyer,  Pyrex* flasks and in 100  oL




polystryene cups with plastic caps.



     If glass test chambers are used,  then they should be acid-stripped




for  ca.  10 tain, in 10 to 12 7, HC1 and rinsed in deionized  water  after




washing.  This removes potentially toxic residues left by the detergent.




If organic compounds have been previously tested in the glassware,  then




a rinse with acetone prior to washing is recommended.
                                 I-
                                    18

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                            TEST CONDITIONS




Temperature. Salinity, Light and Aeration




     The  test  exposure  duration is 2 days followed by a 5  to  7  day




recovery period for the development of. cystocarps.  The exposure temper-




ature  should  be between 22 and 24'C,  and the test salinity should  be




between  28 and 32 4/«0>   For testing receiving waters,  salinity  will




often  be below the desired range and oust be adjusted  with  artificial




sea  salts  (see  SALINITY ADJUSTMENTS).   The photoperiod should  be  a



16h:8h,  light:dark cycle of ca. 75 uE m~  s"  of cool-white fluorescent




light.   It is not necessary for the recovery conditions to be the  same




as the exposure conditions.   However, the conditions listed are optimal




and will result in the fastest cystocarp development.




     Plants  are not aerated during the exposure period.   Chambers  are




either  shaken  at  100 rpm on a rotary shaker or  briefly  hand-swirled




twice a day.   Spermatia are not motile,  therefore some water notion is




critical.  Aeration will inhance the growth rate of plants in the recov-




ery bottles, although, adequate growth will occur using a shaker.






Nutrients




     The  enrichment for natural seawater is listed in  Table  2.   Both




EDTA and trace metals have been omitted.  This recipe should be used for



the 2-day exposure period,  however, it is not critical for the recovery



period.   Since natural seawater quality can vary among laboratories,  a




more complete nutrient medium (e.g.  + EDTA) may result in faster growth



(and therefore faster cystocarp development) during the recovery period.



     The  nutrients  recomended for natural seawater are not  sufficient




for  healthy plants in artificial seawater.   The nutrients for the  GP2




artificial  seawater  are listed in Table 4.   EDTA has act been  elimi-

-------
nated,  but  has been reduced to 20 ug/L  (<17. of the  original  recipe).



One  of the reasons natural seawater is better than artificial  seawater



is  probably  due  to the variety of natural organic  chelators  in  the



former.  Therefore, elimination of all organic chelators from artificial



seawater  should not be necessary.   In fact,  total elimination of EDTA



from  artificial seawater can result in a greater sensitivity  to  toxi-



cants such as copper when compared to results with natural seawater.





Effluent Concentrations



     For end-of-the-pipe samples,  the concentrations that are currently



being used for Champia are 0.63,  1.22,  2.5, 5 and 10 7. effluent plus a



control.  The concentrations recommended in the effluent handling adden-



dum of this Guidance Manual are also acceptable.
                                  1-20

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Table 4.  Recipe of nutrients to be added to GP2 artificial seawater for
stock  cultures and test medium.  See footnotes for differences  between
test  medium  and culture medium.    The concentrated stock solution  is
autoclaved  at  standard temperature and pressure for 15 min (the pH  is
adjusted to 2.0 with HC1 prior to autoclaving to prevent precipitation).
COMPOUND
NaH03
N«H2P04.H20
Na2EDTA.2H20
NaC6H507 (citrate)
Ironc
Trace Elements
tU2«o04.2H20
KI
ZnS04.7H20
N«V03
MnS04.H20
Vitamins
Thiamine-HCl
Biotin
B12
AMOUNT/LITER
STOCK CULTURES CONCENTRATED STOCK
127 mg
12.3 mg
2.66 mgb
1.03 mg
195 ug

24.2 ug
33 ug
21.8 ug
6.1 ug
0.61 ug

1.95 mg
1.0 ug
1.0 ug
12.7 g
1.28 g
266 mg
103 mg
19.5 mg

2.42 mg
3.3 mg
2.18 mg
0.61 mg
61.0 ug


10 mL6

*Use  1.25  mL/L for test medium  (with the adjusted EDTA  concentration)
 and 5 mL/L for stock cultures.   For test media an additional 2.5  mL/L
 of the sodium bicarbonate  stock  solution is added (see Table 3).
      concentration   is   20  ug/L  for  final solution of  test  media  in
 artificial   seawater;  EDTA is omitted entirely  if the test  medium  is
 natural seawater.

cThe  same  stock  solution  as for Table  2.

^Trace  elements are omitted for toxicity test  medium (they  are  also
 generally omitted when this medium is used with  natural seawater) .

Vitamin   stock   solution autoclaved separately in  10  mL  sub-samples.
 Each 10 mL  contains  195  mg of thiamine«HCl,  100 ug biotin,  and 100 ug
 B«2.   Adjust pH to  ca 4.0 before  autoclaving for 2 min.


                                  1-21

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                                PROTOCOL




1.  Set up and label control and treatment chambers; three per treatment




    and controls.




2.  Fill  chambers  with 100 oL of control or treatment water (28 to  30




    */«0).  Alternately,  all chambers can be filled with control  water




    and  the  toxicant  added with micropipets.   For  toxicant  volumes




    exceeding  1  mL,  adjust amount of dilution water to give  a  final




    volume of 100 mL.




3.  Add  the appropriate nutrients and bicarbonate to each chamber  (see




    Table 2 or 4).




4.  Add  five  female branch tips and one male branch to  each  chamber.




    Hake sure the toxicant is present before the male is added.




5.  Place chambers under cool-white light (ca 75 uE m   s" ) at 22 to 24




    *C.  Place a thermometer in a flask of water among the chambers.




6.  Gently  hand-swirl chambers twice a day.   Alternately,  shake  con-




    tinuously at 100 rpm on a rotary shaker.  Record temperature daily.




7.  If desired, media can be changed after one day (24 hr).




3.  After 2 days (43 hr):



    A. Label  recovery  bottles  (these  can  be  almost  any  type   of




       container  or flasks with 200 to 400 oL of natural seawater  plus



       the additional nutrients (see Table 2 or 4).  Smaller volumes can




       be used,  but should be checked to make sure that adequate growth



       will occur without having to change the medium.  As with  culture



       vessels,  all  glassware should be acid-stripped with 10 to 15  7.




       HC1.



    3. With forceps,  gently remove females from test chambers and place




       into  recovery bottles.   Add aeration tubes and foam stoppers.
                                 I-
                                   22

-------
    C. Place  bottles under cool-white light (at the same irradiance  as




       the  stock  cultures) and aerate for  the  5- to  7-day  recovery




       period.   If  recovery  is on a shaker,  then eliminate  aeration




       tubes and reduce the volume of seawater to approximately one-half




       of the vessel volume (this will enhance the water motion).




9.  At the end of the recovery period count the number of cystocarps per




    female and record the data (Addendum C-V).   Cystocarps are  counted




    by  placing  females  between the inverted halves of  a  polystyrene



    petri dish with a small amount of seawater (to hold the entire plant




    in  one  focal plane).   Using  a  stereo-microscope,  the  emergent




    cystocazps can be easily counted*  Cystocarps are distinguished from



    young  branches because they possess an apical ostiole (opening  for




    spore release) and darkly pigmented spores (see Figs.  7,8).  One of




    the advantages to this test procedure is that if there is uncertain-




    ty  about  the identification of an  immature  cystocarp,  then  the




    plants  can  just  be aerated for a little longer  in  the  recovery




    bottles.   Within 24 to 48 hr the structure in question will  either




    look more like a mature cystocarp; look more like a young branch; or




    have  changed  very  little,  if  at all (i.e.,  it  is  an  aborted




    cystocarp).   No new cystocarps will form since the males have  been



    removed,  the plants will only get bigger.  Occasionally, cystocarps




    will  abort,  and  these  should not be included  in  the  cystocarp



    counts.   Aborted  cystocarps  are easily identified by  their  dark




    pigmentation (Fig.  9).   They also often begin to form a new branch




    at their apex.
                                 1-23

-------
                        1mm
Figure 7.   A mature cystocarp of Champla parvula.
     the lower effluent concentrations,  cystocarps
     in groups of as many as 10 to 12.
 In the controls and
are often  clustered
                                  cells
                                 young branch
                                       immature
                                        cystocarp
Figure  3.    Comparison of a very young branch  with  an  immature  cysto-
     carp.   Both can have sterile hairs and the young branch may or may
     not have trichogynes.   However,  the immature  cystocarps will never
     have  trichogynes.    Young branches are usually  more  pointed at the
     apex  and will not  form an ostiole.   The cell dimensions of  young
     branches are larger than those of the cystocarp.
                           1mm
Figure 9.  An aborted cystocarp of  Champia  parvula.
     eventually develop at the apex.
 A new branch  will
                                I-

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                     STATISTICAL TREATMENT OF DATA
     Chronic  values  are calculated using two  different  criteria  for
determining significant differences.   One criterion for difference from
controls  is  absence  of cystocarps;  >_ 957. decrease from  the  control
(Thursby and Steele, 1986).  The other criterion is based on statistical
differences.   Statistically  significant  decreases in  the  number  of
cystocarps  are determined by one-way analysis of variance (ANOVA)  fol-
lowed by Dunnett's mean separation test (alpha * 0.05) for comparison of
treatments  with a control (Steel and Torrey,  1960).   The results from
each replicate chamber are reported as the mean number of cystocarps per
plant (n«3 for each treatment).
     Chronic  values are expressed either as the  no-effect  range,  the
lowest  concentration that results in a significant difference from  the
control  and the next lowest concentration tested,  or as the  geometric
mean of these two values (Buikema, et als j—1982).  If the lowest coneen-
                      f
tration  that results in a significant difference is the lowest  concen-
tration tested, then the geometric mean is not calculated.  One can only
report  the  lowest concentration that gave the  significant  difference
(one  should  also consider repeating  this  test).   In  practice,  the
"absence  of cystocarps" endpoint is generally used for determining  the
effect concentration from an effluent diluton series.   However,  if the
concentration  that  causes a statistical decrease from the  control  is
also  reported,  then  some idea of the steepness of the  dose  response
curve  can be inferred.   The statistical difference is  used  primarily
when testing receiving waters where dilutions are usually not made.
                                 I-
                                   25

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                       CRITERIA FOR ACCEPTABILITY




1.  A test is not acceptable if control mortality exceeds 207.  (generally




    there is no control mortality).




2.  If  plants  fragment in either the controls or the  lowest  exposure




    concentration so that individual plants can not be identified,  then




    the test is not acceptable.   This is aot critical if absence of sex




    is the only endpoint of interest.  However, the fact that  the plants




    fragmented  indicates they are not at their best and the data may be



    biased toward the lower concentrations.




3.  A  test should not be considered definitive if the controls  average




    fewer  than  10 cystocarps per plant.   If  no  sexual  reproduction




    occurs in the controls, then this test can not be considered accept-




    able.




4.  If  the  plants  in the two control chambers  are  suspected  of




    responding differently (this can be checked with a t-test), then the




    test should not be considered acceptable.



5.  The  data  from all replicates at the effluent concentration  deter-




    mined to be the effect concentration should be statistically  equal.




    That is, all replicates should show the effect.
                                 1-26

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                               REFERENCES




Buikema A.L.,  Niederlehner B.R.,  Cairns J.  1982.  Biological monitor-




     ing, Part IV,  Toxicity Testing.  Water Res. 16:239-262.




Jensen A.  1984a.  Marine ecotoxicological tests with seaweeds, pp. 181-




     193.   In:  Ecotoxicological  Testing for the  Marine  Environment.




     Persoone G.,  Jaspers E.,  Claus C.  (eds).  State Oniv.  Ghent and




     Inst. Mar. Sclent. Res., Bredene, Belgium. 7ol 1.




Jensen A.   1984b. Marine Ecotoxicological Tests with Phytoplankton. pp.



     195-213. ibid.




Kenaga E.E. 1982. The use of environmental toxicology and chemistry data




     in  hazard  assessment:   Progress,  needs,  challenges.   Environ.




     Toxicol. Chem.  1:69-79.




Kenaga  E.E.,  Moolenaar  R.J.   1976.   Fish  and Daphnia  toxicity  as




     surrogates for aquatic vascular plants and  algae.   Environ.  Sci.




     Techno1. 13:1479-1480.




Spotte S.,  Adams 6.,  Bubucis P.M.   1984.  GP2 medium is an artificial




     seawater  for  culture or maintenance of  marine  organisms.   Zoo.




     Biol. 3:229-240.




Steel R.G.,  Torrey J.H.   1960.   Princilples and procedures of statis-




     tics.  McGraw Hill.  481 pp.



Steele  R.L.,  Hanisak  M.O.   1978.  Sensitivity  of some  brown  algal



     reproductive stages to oil pollution.  In: Proceedings of the Ninth



     International Seaweed Symposium, pp. 181-190. Jensen A., Stein J.R.




     (eds). Science Press.



Steele R.L.,  Thursby G.B.   1983.  A toxicity test using life stages of



     Champia parvula (Rhodophyta).   In:  Aquatic Toxicology and  Hazard



     Assessment,  Sixth Symposium,   pp.  73-89. Bishop WC, Cardwell RD,



     Heidolph BB (eds).  ASTM STP 802.   American Society of Testing and






                                 1-27

-------
     Materials, Philadelphia.



Thuzsby G.B.,  Steele R.L.   1984.  Toxicity of arsenite and arsenate to



     the  marine  macroalga  Champia  pannila   (Rhodophyta).   Environ.




     Toxicol. Chen. 3:391-397.



Thursby G.B., Steele R.L., Kane M.E.  1985. Effects of organic chemicals



     on  growth and reproduction in the marine red alga Champia parvula.



     Environ. Toxicol. Chem. 4:797-805.



Thursby,  G.B.,  Steele, R.L.  1986.  Comparison of short- and long-term



     sexual reproduction tests using Champia parvula.  Environ. Toxicol.




     Chem. (in press).
                                  1-28

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                              ADDENDUM C-I

List of materials for performing toxicity test3 with Champia parvula for
effluent program.
 1.  Fine-point stainless steel forceps.

 2.  100 mL polypropylene cups with covers (or 125 mL Erlenmeyer flasks)
     to be used as exposure chamabers.

 3.  Polystrene  petri dishes to hold plants for counting cystocarps and
     for cutting branch tips.

 4.  100 mL graduated cylinder to measure control and treatment volumes.

 5.  1 and 10 mL disposable pipets.

 6.  Digital micropipets (200 and 1000 uL maxima)  if dilutions are made
     directly in test chambers.

 7.  Disposable tips for micropipets.

 3.  Nutrient and sodium bicarbonate stock solutions.

 9.  Recovery  bottles  or flasks,  need one per  treatment  or  control
     chamber.

10.  Aquarium pump(s) and air tubing.

11.  Plastic  aeration tubes (1 mL disposable pipets work fine) and foam
     plugs.

12.  Thermometer and flask or bottle to hold it.

13.  Marking pens and colored tape.

14.  Cool-white fluorescent, lighting,  sufficient to give 75 uE m~   s"
     (ca 500 foot-candles).

15.  Rotary  shaker for exposure chambers (hand-swirling twice a day can
     be substituted).

16.  Stereomicroscope for counting cystocarps.

17.  Refractometer for salinity measurements.

18.  Data sheets (one per test).

19.  Protocol.
                                 I- 29

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                             ADDENDUM Oil

Summary of test conditions for Champia parvula sexual reproduction test,
1.  Test type:

2.  Salinity:

3.  Temperature:

4.  Photoperiod:

5.  Light source:

6.  Irradiance:

7.  Test solution volume:

3.  Test chamber size:
9.  Number of test organisms
     per test chamber:

10. Number of replicate
     chambers per treatment:

11. Aeration:
12. Dilution water:
13. Test duration:
14. Effect measured:
Static, non-renewal

30V,,

22 to 24 «C

16h light:8h dark

cool-white fluorescent

ca 75 uE m"2 s"1

100 oL

110 mL polypropylene cups (with
covers) or 125 mL Erlenmeyer flasks

5 female branch tips and one male
None; chambers are either shaken at
100 rpm on a rotary shaker or hand-
swirled twice a day

30 Va, natural or artificial
seawater with additional nutrients
added

2 day exposure followed by a 5- to
7-day recovery period for cystocarp
development

Sexual reproduction (number of
cystocarps per female)
                                 1-30

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                             ADDENDUM C-III

Equations for making salinity adjustments.

     A.  To dilute to a desired salinity with deionized water.

                           Six7i-Sfx7f

          where:

          S  » initial salinity (measured)

          Sf • final salinity (selected)

          V  * initial volume (unknown)

          7. - final volume (selected)

Solve for V.,  then dilute to the final (selected) volume with deionized
water.


     B.  To mix two different salinities to get a third salinity.


                   si(vf - V * sn * 7n - sf x 7f
                                  and
                             7  » 7  +
                             Vf   VI
          where:
          S_ * salinity of the first solution (measured)

          S__ • salinity of the second solution (measured)

          S. • salinity of the final solution (selected)

          7_ - volume of the first solution (unknown)

          7_T » volume of the second solution (unknown)

          V, * volume of the final solution (selected)

Solve the first equation for 7  , then solve the second equation for


Example:  Solution I salinity » 10 a/ao;
          solution II salinity - 90 °/«a;
          final solution salinity desired « 30- a/00;
          final volume wanted « 1000 mL.
                                 1-31

-------
First equation becomes: 10 (1000 - 7  ) + 90 7 _ » 30 x 1000

                        10,000 - 10 V   + 90 VTI - 30,000

                                          80 Vjj - 20,000

                                             7   - 250 mL
Second equation becomes:                      7. > 1000 - 250

                                              7  - 750 mL
Any  of the three volumes can be selected as the constant,  solving  for
the other two volumes.   For example you may have 500 mL of solution  II
and wish to know how much of solution I to add to get 30 */a«.   In this
case  you  would just solve the first equation for 7. and continue  from
there .
                                 I- 32

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                             ADDENDUM C-IV

Precision testing for Champia parvula short-term.

     Intralaboratory precision testing of the short-term exposure repro-
ductive test with Champia parvula has not been conducted yet.   However,
we  have  several repeat tests with effluents and  one  single  compound
(copper).   All tests concentrations were unmeasured.  The concentration
given  is the geometric mean of the effect/no-effect level.   Effect  is
defined as having any number of cystocarps/plant £ 51. of the control. In
general,  the  agreement among the repeat tests was very good.   For the
heavy metal effluent,  the variation was by a factor of 3,  but the  two
tests (on the same sample) were run approximately 2 months apart.
COMPOUND
COLLECTION DATE
TEST DATE
Heavy Metal
  Effluent
Organic Effluent
    8/17/84
    8/17/84
                     5/8/85
                     5/8/85
EFFECT LEVEL
Copper




2/18/85
2/18/85
4/3/85
4/3/85
6/7/85
~ 8.8 ug/L
8.8
8.8
8.8
8.8
 9/25/84           1.76 I
11/27/84           0.56
                     5/16-shaken      14.1 7.
                     5/16-not shaken  14.1
                                  1-33

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ADDENDUM C-V
    1-34

-------
                   CHAMP/A   PARVULA
                   CYSTOCARP  DATA  SHEET
COLLECTION DATE
EXPOSURE BEGAN (date).
RECOVERY BEGAN (date),
COUNTED (date) 	
EFFLUENT OR TOXICANT
              TREATMENTS (% EFFLUENT, ^/G/L,  or REC. WATER SITES)
REPLICATES

A 1
2
3
4
5
CONTROL







MEAN





















































9 1
2
3
4
3



- -:


MEAN











































c ^
2
3
4
5

MEAN

OVERALL
MEAN






























































  Temperature.
  Salinity ——
  Light 	
  Source of Dilution  Water.

-------
       RECEIVING  WATER SUMMARY  SHEET
       SITE
       COLLECTION DATE
       TEST DATE 	
LOCATION










INITIAL
SALINITY










FINAL
SALINITY










SOURCE OF SALTS FOR
SALINITY ADJUSTMENT *










* .
 i.e. natural seawoter brine, GP2 brine, GP2 salts , etc.
(include some indication of amount)
 COMMENTS:

-------
                          PART II
COMPARISON OF SHORT- AND LONG-TERM SEXUAL REPRODUCTION TESTS




          WITH THE MARINE RED ALGA CHAMPIA PARVULA
                               II-l

-------
Abatract--A  two-day  exposure  test using the marine red  alga  Champia



parvula  has been developed for assessing the toxic effects  of  complex



effluents  entering the marine and estuarine environments.   The initial



exposure  was  followed by a 5- to 7-day recovery period  to  allow  the



development  of  any  cystocarps (evidence of  sexual  reproduction—the



endpoint measured).  The two-day exposure test was validated by compari-



son with a previously developed two-week test in which "no sexual repro-



duction" (NSR) was used as the reproductive endpoint  measured.   Single



compounds can be more accurately tested with the two-week test procedure



than effluents,  therefore,  they were used to compare the two-day expo-



sure test with the two-week exposure.   A total of nine single compounds



were  tested  using the two  testing  procedures.   Concentrations  that



resulted in a 95H or greater decrease from the control response with the



two-day  exposure were considered the best estimate of the NSR  response



from  the  two-week test.   All of the single compounds  tested,  except



cadmium, yielded essentially the same results with both tests.
Keywords--Champia, effluents, sexual reproduction
                                 II-2

-------
                              DTTBODUCnOM

     The Permits Division of the U.S.  Environmental Protection Agency's

(EPA) Office of Water needs toxicity test methods for marine and  estua-

rine  species.   The  U.S.  EPA'a Environmental Research  Laboratory  at

Narragansett, Rhode Island has developed or modified four toxicity tests

to begin to address the above need.   The four species used are: the red

macroalga,  Champia parvula;  the sea urchin,  Arbacia  punctulata;  the

my3id,   Myaidopsis  bahia;   and  the  sheepshead  minnow,   Cyprinodon

veriegatus.  The  methods  are  needed to characterize the  toxicity  of

effluent discharges within the National Pollutant Discharge  Elimination

System  (NPDES).   Since  March 9,  1984,  EPA has had the authority  to

require biological testing as a condition for issuing HPDES permits [!]•

These tests should yield chronic data in a relatively short time  period

(7  days or less).   It is also necessary that the methods be simple and

cost efficient (requiring standard hardware and laboratory  facilities).
                 •>
The species used should be readily available, and should also be practi-

cal for both on-site and off-site testing.   All of the requirements are

met by the test method for the marine red macroalga, Champia parvula.

     Seaweeds have been considered-less useful for toxicity testing than

microalgae [2],  and microalgae are often considered less sensitive than

aquatic animals [3,4].   Therefore,  one could easily come to the erron-

eous  conclusion  that toxicity testing with seaweeds is not  necessary.

Recently the statement was made that "seaweeds seem to be rather  insen-

sitive to many chemicals and will probably survive pollution better that

many other organisms in the marine environment" [2].

     Previous conclusions about seaweed sensitivity were based on only a

few  species,  and  generally considered only vegetative growth  of  the
                               II-3

-------
macroscopic  life  history stage as the endpoint.   The  sensitivity  of



seaweeds  can increase when sexual reproduction is used as an  endpoint,



and   can  be greater than many aquatic animals that have  been  tested.



This has been shown for the red alga,  Champia parvula [5-7], as well as



with  the brown algae Fucus edentatus and Laninaria saccharina  [8].  In



addition,  using  growth of young sporophytes,  ^  saccharina has  been



placed among the most sensitive marine organisms for toxicity of copper,



zinc and mercury [9].



     A  two-week toxicity test has already been developed for the macro-



alga, Champia parvula, to assess chronic effects of pollutants to marine



seaweeds  [5].   The  test  has been evaluated with  heavy  metals  [5],



arsenite  and  arsenate  [6] and ten different  organic  compounds  [7].



Sexual  reproduction  was  generally the most  sensitive  and  practical



endpoint to use for C.  parvula.   The two-week exposure,  however,  made



this test procedure unacceptable for testing complex effluents and their



receiving waters.'



     Effluents  can  not  be  easily  tested  using  the  two-week  test



procedure.  The two-week toxicty test with Champia parvula requires  that



the cultures remain unialgal during the test period.   Fine-filtering or



autoelaving,  which would be necessary to eliminate unwanted microalgae,



could  change  the character of the effluent or  receiving  water.   Any



microalgae  introduced with the effluent would compete with  C._  parvula



for  light and nutrients,  thus influencing^  parvula's  growth  rate.



However,  if  sexual  reproduction is used as the  sole  endpoint,  then



plants  only  need  to be exposed for a few days (long  enough  to  show



effects on fertilization).   Any effect of other organisms on the growth



rate of C. parvula should not be serious since interest would only be in



whether  sexual  reproduction had taken place.   The two-week  procedure





                               II-4

-------
also  requires that the media be changed during  the  test.    Therefore,



either  the effluent sample would have to be stored (i.e.  refrigerated)



or additional samples would have to be collected during the  test period.



Both  of these alternatives would result in variable  toxicity.    Single



compounds can be more accurately tested with the two-week  test procedure



than  effluents*  therefore,  they  were  used to  compare  the   two-day



exposure test with the two-week exposure.



     This  paper describes a method which uses sexual  reproduction   to



estimate chronic toxicity  after a short-term exposure to  toxicants.   In



brief, the method consists of exposing males and females to  effluents  or



receiving waters for two days, followed by a 5- to 7-day recovery period



in  control medium.   The recovery period allows time for  any cystocarps



to mature.   At the end of the recovery period the number  of  cystocarps



per plant are counted.
                              II-5

-------
                         MATERIALS AHD METHODS



Maintenance of stock cultures
     Unialgal  stock  cultures of both male and female  gametophytes  of



Champia parvula (C.  Agardh) Harvey were maintained in separate, aerated



1000  mL  Erlenmeyer flasks containing 800 mL of  culture  medium.   All



culture  glassware  was  acid-stripped in 10 to 15 % HC1 and  rinsed  in



deionized water after washing.  The culture medium was made from natural



seawater (from lover Narragansett Bay, RI) to which additional nutrients



were  added (Table 1).   The seawater was filtered through a 15-urn char-



coal filter and a 0.3-um Balston filter,  then autoelaved for 30 min  at



15  pai in 20 L carboys.   The culture flasks were capped with  aluminum



foil  and autoelaved dry for 10 min.   Culture medium was formulated  by



dispensing  seawater into the sterile flasks and adding the  appropriate



nutrients from a sterile stock solution.



     Initial stock cultures were started weekly with about twenty 0.5 to

                  »

1.0  cm  branch tips.   Cultures were gently  aerated  through  sterile,



cotton-plugged,  disposable,  polystyrene  1 mL pipets.   Cultures  were



capped  with foam plugs and aluminum foil and illuminated from the  aide


               •2   -1
with  75  uE  m    s    of cool-white fluorescent  light  on  a  16h:8h,



light:dark cycle.   The temperature was 22 to 24 °C and the salinity  28



to 30 °/«0*  Media were changed once each week.



     About  half  of the plants were discarded (or placed  into  another



culture  vessel) with each weekly medium change to reduce the amount  of



biomass as the plants grew.  At the end of three weeks plants were ready



to  use  for  testing.   Readiness was defined as  having  enough  plant



material to perform at least one test.   With this  procedure,  actively



growing plants were continuously available.
                                 II-6

-------
     A  stock  culture was not used as a source of test material if  the



plants appeared to be stressed or undernourished.   Under conditions  of



stress  the tips of the branches turned "pink" and the older tissue  was



generally   much  paler.    Under  conditions  of  nutrient   deficiency



(resulting  usually from too much plant material in the culture flask or



too  long  since the last medium change) the entire  plant  turned  pale



yellow.   If  the stress was severe enough the older tissues (main axes)



or  occasionally  the  branch tips turned white  (evidence  of  necrotic



tissue).






Two-week test



     All  procedures  for  the two-week test followed  those  previously



described [5], except EDTA was omitted from the medium and vitamins were



added.  The test medium was then identical  to that used for the two-day



exposure  test.   Toxicity test duration was 14 days.   Tests were  per-



formed  with 400 oL volumes in 500 mL,  screw-capped Erlenmeyer  flasks.



The  medium  was  fc strength of that used for the  stock  cultures.   In



addition  150  mg/L sodium bicarbonate was added rather  than  aeration.



Flasks were shaken on a rotary shaker at 100 rpm.   Media were  replaced



on  days  7,  and 11.   All other conditions were the same as those  for



stock cultures.



     After  stock  cultures were rinsed in sterile  seawater  to  remove



traces  of old medium,  2- to 3-mm branch tips were cut from females  to



serve as inocula for toxicity tests.   Five branch tips were placed into



each test flask.   One male branch (about 1 cm long),  visibly producing



spermatia, was added to each flask containing females.  Replicate flasks



were used for each treatment.  At the termination of each toxicity test,



females were examined for the presence of cystocarps (evidence of sexual
                                II-7

-------
reproduction).   The concentration which resulted in no sexual reproduc-



tion (HSR) was determined.






Two-day exposure



     Stock  cultures were rinsed in sterile seawater to remove traces  of



old medium.  Female branch tips, 7 to 10 mm in length,  were cut to serve



as inocula.   Five tips were placed into each treatment cup,  along with



one  male  branch (1.5 to 2.0 cm  long),  visibly  producing  spermatia.



Tests were performed in replicate 100 mL polystyrene cups,   with plastic



caps,  containing 80 mL of medium.   The nutrient medium was the same  as



for the two-week tests.



     The two day exposure was followed by a5- to 7- day recovery period



(for  females  only)  in  control  medium   for  cystocarp   development.



Temperature,  salinity  and light conditions were the same  as for  stock



cultures,  except  that light was from above.   Plants  were not  aerated



during the exposure period.  Exposure chambers were shaken  at 100 rpm  on



a rotary shaker.  Recovery bottles were aerated, since  this enhanced the



growth  rate  of  plants  and  therefore  the  rate  of  development  of



cystocarps.



     At  the  end of the recovery period the number  of  cystocarps per



female were counted.   The results from each replicate  cup  were reported



as  the  mean number of cystocarps per plant (n » 2 for the  treatment).



The data were examined for HSR,  95 and  507. decreases  from the control,



and statistical differences from the control.   Statistical decreases  in



the number of cystocarps were determined by one-way analysis of variance



(ANOVA)  followed by Dunnett's means separation test (alpha - 0.05) for



comparisons of treatments with a control [10].



     One  advantage  of this test procedure is if there  is  uncertainty
                               II-8

-------
about  the identification of an immature cystocarp,   then the plants can



Just be aerated a little longer in the recovery bottles.    No new cysto-



carps  will  form since the male gametophytes  have   been  removed;   the



plants will only get bigger.



Toxicant concentrations



     The   highest   concentration  tested  was  based on   preliminary



experiments and was chosen to cause death or a near  death response after



a  two-week  exposure.  The dilution factor for all  test  runs  was  0.6.



Only   one  stock  solution  was  prepared  for  each  toxicant.     All



concentrations  were obtained by dispensing from these stock  solutions



with adjustable micropipets.



     Stock  solutions  of  sodium  arsenite,   copper  sulfate,  cadmium



chloride and silver nitrate were prepared in deionized water.  Toxaphene



was  dissolved first in acetone and then diluted with triethylene glycol



(TEG)  to give a final acetone to TEG ratio of 1  to  20.   Pentachloro-



ethane  and pentachlorophenol were prepared in TEG alone.   Benzene   and



isophorone were dispensed directly.   For benzene, isophorone and penta-



chloroethane (all liquids),  density was used to calculate the weight of



the compound in solution.   A carrier control (at the highest concentra-



tion of carrier used with a toxicant) was used with  those toxicants  that



required a carrier for solubility.  Water samples from the test chambers



were not chemically analyzed for toxicant concentrations,  therefore all



concentrations reported are nominal concentrations added.
                                II-9

-------
                         RESULTS AMD DISCUSSION



     The results of the comparison of the two-day exposure test with the



two-week  test using single compounds are shown in Table 2.   Only  data



for concentrations causing no sexual reproduction (HSR) are reported for



the  two-week test.   Several values are listed for the two-day exposure



test.   The  object of the two-day exposure was to estimate the  chronic



effect from the two-week test.   The endpoint of choice for the two-week



test  was NSR,  therefore all two-day results are compared only to  this



number.   The  same concentrations were tested in both test  procedures,



and for four of the nine compounds the NSR endpoint was not achieved for



the  two-day test.



     The  best  two-day values for estimating the two-week  NSR  results



were  those  from  the concentration that resulted in a 95%  or  greater



decrease in the number of cystocarps (0 to 5% of control) when  compared



to  the controls.  This is seen more clearly in Table 3 which shows  the



ratios of the values in Table 2 to the two-week values.  Cadmium was the



only  compound tested that did not give a good relationship between  the



two-day and the two-week  results.    Cadmium is generally a slow acting



toxicant  [11,  12] and two days may not have been enough time to elicit



its effect.   The ratios for arsenite and silver are close to one, while



the remaining are one.   Arsenite and silver were the only two compounds



for which the two test procedures were not started on the same date.  It



should  be noted that the two-day benzene test was performed in 400  mL,



screw-capped  Erlenmeyer flasks instead of the polystyrene  cups.   When



plants  were exposed in 80 mL test solution the toxicity was much  less,



probably due to the high volatility of benzene.   If volatile  compounds



are being tested,  then larger, air-tight exposure chambers should prob-



ably  be  used.  However,  the  two day test procedure is  designed  for
                            11-10

-------
testing effluents and receiving waters.   Highly volatile compounds  are


significantly  reduced in pretreatment before effluents are  discharged.


Therefore the failure of the test with benzene when using smaller,  non-


air-tight  vessels  is  not expected to be a problem  when  testing  ef-


fluents .


     A  sample  data sat is graphed for pentachloroethane in  Figure  1,


illustrating  that a 95% decrease from the control is a good  value  for


estimating the two-week results.   The dose-response curves for both the


two-week  test and the short-term exposure tests are similar.   However,


no cystocarps were produced during the two-week test at 13,000 or 22,000


jug  L* ,  whereas a few were produced at both concentrations during  the


short-term test.  A similar relationship between the two-day an the two-


week results was seen for most of the compounds tested.


     Sexual  reproduction was selected as the endpoint for two-day test-


ing for several reasons.  It was proven previously to be a sensitive and
                »

practical  endpoint from the two-week toxicity test procedure  [5-7].  A


sexual  reproduction test for toxicity could be short enough to fit  the


time  constraints for tests used in the effluent program (ca.  7  days).


Finally, Champia parvula is an annual plant and inhibition or absence of


sexual  reproduction  reduces or eliminates the next stage in  the  life


history.   Total  absence of cystocarp formation is the easiest endpoint


to interpret as far as field populations are concerned.   In most of the


red  algae,  each fertilization results in the formation of a  new  life


history stage, the earposporophyte, "parasitic" on the female and housed


within the cystocarp.  Each carposporophyte is capable of producing many


spores  (perhaps  a hundred or more in the case of  C.  parvula).   This


characteristic  makes it difficult to interpret the  biological  signif-
                                  11-11

-------
Icance  of  a  statistical decrease in the number of  cystocarps  or  an



arbitrary percent decrease such as 501.   Absence of reproduction leaves



no doubt about its biological significance.



     The  two-day  exposure  test has been used successfully in  both  a



mobile  laboratory  and  in  the  main  laboratory  (EPA,  Environmental



Research Laboratory,  Narragansett,  RI,  USA).   The method is easy and



coat-efficient to perform.  Stock cultures are maintained in the labora-



tory with standard laboratory equipment,  therefore,  plant material can



be available throughout the year.  The test procedure la intended to  be



used  to estimate chronic effects of complex effluents on marine  macro-



algae,  although  it  can obviously be used for single  compounds.   The



procedure  has  already  been included in a draft  guidance  manual  for



tasting marine and estuarine effluents.  Comparing this test with a two-



week chronic teat using single compounds has shown  that the test can be



uaed  for  determining  adverse effects on sexual  reproduction  in  the



marine alga, Champia parvula.








Acknowledgements—The  technical  assistance  of  Raymond  Palmquist  is



greatly appreciated.
                                  11-12

-------
                              UFfiUKBO
1.  U.S.  Bmjjiuuaeutal Protection Agency.  1984.  Development of water




    quality-baaed  permit limitations for toxic  pollutants:   National



    policy.  Fed. Reg. Vol. 49 No. 48. Friday, March 9, 1984.



2.  Jensen,  A.   1984.   Marine ecotoxicological tests with  seaweeds,




    In G.Persoone,  E.  Jaspers and C.  Glaus,  eds.,  Ecotoxieologieal



    Testing for the Marine Environment,  Vol.  1. State Univ. Ghent and



    Znst. Mar. Scient. Res., Bredene, Belgium, pp. 181-193.



3.  g*"*tr,  K.K. and R.J* Moolenaax.  1976.  Fish and Daphnia toxicity



    as surrogates for aquatic vascular plants and algae.  Environ. Sci.



    Techno1. 13*1479-1480.
4.  g^"*tTi  K«Ea   1982.   The  use of  environmental  toxicology  and



    chemistry data in hazard assessment:   Progress, needs, challenges.



    Environ. Toxieol. Chem. 1:69-79.



5.  Stewla,  R.L. and G.B.  Thnraby.   1983. A toxicity test using life



    stages  of  Champia parvula  (Rhodophyta).  In  V.E.  Bishop,  R.D.



    Cardwell  and B.B.  Heidolph,  eds.,  Aquatic Toxicology and Hazard



    Assessment:   Sixth Symposium.   ASTM STP 802. American Society for



    Testing and Materials, Philadelphia, PA, pp. 73-89.



6.  Tharsbr,  G.B. and R.L.  Steele.   1984.  Toxicity of arsenite  and



    arsenate  to  the  marine macrolaga Champia  parvula  (Rhodophyta).



    Environ. Toxieol. Chem. 3:391-397.



7.  Tharabr,  G.B., R.L. Steole and H.E. Kane.  1985. Effect of organic



    chemicals on growth and reproduction in the marine red alga Champia



    oarvula.  Environ. Toxieol. Chem. 4:797-805.
                                 II
                                   -13

-------
 8.  Steele,  R.L. and M.D.  Hanisak.   1978.  Sensitivity of some brown



     algal reproductive stages to oil pollution.  In A.  Jensen and J.R.



     Stein,   «ds.,  Proceedings  £f_  the  Ninth  International  Seaweed



     Symposium, Vol. 9, pp. 181-190.




 9.  Tbo«B«o»,  R.S. and S.M.  BOTTOM.   1984.  The toxicity of copper,



     zinc,  and mercury to the brown macroalga Laminaria saccharine,  In



     G.Persoone, E. Jaspers and C. Glaus, ads., Ecotoxicological Testing



     for the Marine Environment,  Vol.  2.  State Univ.  Ghent and last.



     Mar. Sclent. Res., Bredene, Belgium, pp. 259-269.



10.  Steel,  R.6* and J.H.  Terries.   1960. Principles and Procedures £f_



     Statistics. McGraw-Hill, New York, NT



11.  lagexaoil,  C.G« and R.L. Winner. 1982. The effect on Daphnia pulex



     of  daily pulse exposure to copper or  cadmium.  Environ.  Toxicol.




     Chem. 1)321-327.



12.  Arillo,  A.,  D.  CalasMxi,  C. Margiocco, P. Melodla and P. Meaai.



     1984.   Biochemical  effects  of long-term exposure to cadmium  and



     copper  on  rainbow trout (Salmo gairdneri):  Validation  of  water



     quality criteria. Ecotoxico1. Environ. Safety 8>106-117.
                                    11-14

-------
Table  1.   Recipe fox additional nutrients to be added to natural  sea-



water  for Champia parvula stock cultures and test medium.    Both  EDTA



and  trace metals have been omitted.  The concentrated stock solution is



autoclaved at standard temperature and pressure for 15 minutes.
COMPOUND
NaN03
NaHjPO^HjO
Ironb
Vitamins
B12
Biotin
Thiaaln««HCl

TEST MEDIUM C
9.35 mg
0.62 mg
2.6 ug

0.06 ug
0.06 ug
12.5 ug

AMOUNT/LITER
ONC. CONCENTRATED STOCK*
3.74 grams
0.25 grams
1.04 mg


10 mLe


*Use  0.2 oL/SOmL (2.5 mL/L) for test medium concentrations and 10  mL/L



 for stock cultures.   For test medium only, add 0.2 mL/80mL of a sodium



 bicarbonate solution.   A stock solution of 60 mg/mL sodium bicarbonate



 is prepared by autoclaving it as a dry powder and then dissolving it in



 sterile deionized water.



 Iron  stock  solution prepared by dissolving 1 g iron powder in  10  mL



 concentrated HC1 plus ca.  1 mL of deionized water.  This is diluted to



 1  liter with deionized water.   Acceptable stock solutions can also be



 made with ferric or ferrous chloride.



Vitamin  stock  solution autoclaved (2 min) separately in  10  mL  sub-



 samples.   Each  10  mL  contains  24 ug B12,  24 ug biotin  and  5  mg



 thiamine*HCl.  Adjust pH to ca. 4.0 before autocalving.
                               11-15

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Table 2. Test results for Champla parvula comparing the two-day exposure



test  and the two-week test using single compounds.   Values listed  are



the  geometric  means of the effect and the no-effect concentrations  in



ug/L.   HSR  refers to no sexual reproduction,  i.e.  no cystocarps were



formed  at  these concentrations.   The other column headings  refer  to



concentrations that resulted in 95 and 501 decreases from controls,  and



concentrations  that  resulted in a number of  cystocarps  statistically



less than controls.
COMPOUND
Arsenite*
Copper
Q4HJm^ni»
Silver*
b
Benzene
Pentachloro-
e thane
Pentachloro-
phenol
Toxaphene
Isophorone
NSR
>300
7.7
>100
1.5
7 3 ,.600
> 21,800
465
140
> 138, 500
TWO-DAT
951
230
4.6
>100
0.9
73,600
10,200
465
140
107,300
EXPOSURE
501
84
1.0
>100
0.9
< 34, 3 00
2,200
280
84
38,300
Stat. Dlf.
84
<0.8
17
0.5
44,250
<1,700
280
84
38,300
TWO-WEEK
NSR
140
4.6
77
1.5
73,600
10,200
465
140
107,300
aShort-term exposure and two-week teat not run silmultaneously.



 Both short-term exposure and two-week test run in 400 mL volume.
                                11-16

-------
Table  3.   Ratio of two-day exposure results and two-week test  results



from Table 2 for Champia parvula.  See Table 2 for explanation of column



headings.






COMPOUND                      HSR     57.     501     Stat.  Dif.






Arsenite                      >2.1    1.6    0.6        0.6



Copper                         1.8    1.0    0.2       <0.2




Cadmium                       >1.3   >1.3   >1.3        0.2



Silver                         1.0    0.6    0.6        0.3



Benzene                        1.0    1.0   <0.5        0.6



Pentachloroethane             >2.1    1.0    0.2       <0.2



Pentaehlorophenol              1.0    1.0    0.6        0.6



Toxaphene                      1.0    1.0    0.6        0.6




Isophorone                    >1.3    1.0    0.4        0.4
                                 11-17

-------
Figure 1.  Comparison of the two-day exposure test results and the  two-



week  teat  results for Champia parvula  using  pentachloroethane.   The



graph  illustrates the use of the 95% decrease as the  cut off point for



the two-day exposure test.   Note that some cystocarps were produced  at



the  two  highest concentrations during the two-day  exposure,  but  not



during the two-week test.
                                  11-18

-------
  IOOC
                 O TWO WEEK TEST


                 • TWO DAY EXPOSURE
O
o:
O  50
O


vP
    0
         PENTACHLOROETHANE (x!0//g/L)

-------
                    PART III
SUMMARY OF RESULTS FROM TESTING COMPLEX EFFLUENTS

-------
INTRODUCTION




     Tests  have  been conducted on-site in a  mobile  laboratory  and  at




the EPA1s Environmental Research Laboratory,   Narragansett, RI.  Several




different  types of complex effluents have been tested over the past two




years.   These  include  one  from a pulp mill;   two industrial  sites




that discharge effluents containing heavy metals;   five  industrial sites




discharging organically contaminated effluents, including pestcides and




dyes;  and 17 different sewage effluents*   In all,  over 100  tests were



conducted.




     More complete data is included for the pulp mill effluent (Tables 2




and  3,  and  Figures  1 and 2) from ITT Rayonier   in Fernadina  Beach,




Florida.   These  results  may  be incorporated into  the  first  marine



toxicity-based NPDES permit.






MATERIALS AND METHODS



     The test procedure used is described in the guidance manual (Part I




of  this report).   In brief,  the method consists of exposing males and




females  to effluents or receiving waters for  two  days,  followed  by  a



five- to  seven-day  recovery  period in control  medium.  The recovery



period  allows  time for any cystocarps to mature.   At  the end  of  the




recovery period the number of cystocarps per plant are counted.  Some of



the  earliest tests differed in that the exposure  period was   four  days




instead  of the now standard two days.   However,   our earlier work also



showed  that  two  and four day exposures yielded  essentially  the  same




results.



     Chronic  values can be calculated using two different criteria  for



determining significant differences.   One criterion  for difference from



controls is absence of cystocarps;> 957. decrease from the control.   The
                                 III-2

-------
other criterion is based on statistical differences.  See Parts I and II

of this report for a further explanation.


RESULTS

     Table 1 summarizes all of the effluent test results to  date.   The

summary  is  based on absence of reproduction as the measurement of  the

effect.   In  general the effects could be  separated based  on  effluent

type.   Heavy  metal  containing effluents  were  by  far  the  most  toxic,

followed by oranic and pulp mill effluents,  and  then sewage effluents.

     Tests on the pulp mill effluent from ITT Rayonier  were performed on

four separate effluent and receiving water  collections  (May 15,  17,  18

and 19,  1986).   The tests set up on May 15th and  19th are not included

in these results.  The control values from  these two runs were unaccept-

able  (an average of <10 cystocarps per plant).   The May 15th run  used

plants cultured in artificial seawater, these plants are more difficult

to  judge as ready-to-use in the absence of  a compound  microscope.   The
                   *
test  runs on the other three days all used plants  cultured  in  natural

seawater (where color of the tissue is a good indicator of "readiness").

However,  the test run set up on the 19th lasted only one day instead of

the usual two.

     Tables 3 and 4 list the data from the  May 17th and 18th collections

for the effluent and receiving waters respectively.  Figure 1 is a graph

of  the May 18th data against percent effluent,   based  on  the  previous

week's   dye   study.    These   results indicated that   the   effect

concentrations   in  the  receiving  water   was   consistent  with   that

determined  in the effluent test.   Figure  2 is  a graph of the May  17th

and 18th effluent data and data from an ammonium chloride toxicity  test

run  started on June 11,  1986.   The similarity between the two  curves
                                         III-3

-------
suggests  that  ammonia  may be the main toxicant in   the   ITT  Rayonier



effluent that inhibits sexual reproduction in Champia.   This conclusion



supports  the chemical fractionation studies conducted on-site by  EPA's



Duluth  laboratory  inwhich  unionized  ammonia was   the  primary  toxic



comnponent of the effluent.
                                 III-4

-------
Table  1.   Summary of the results for all the  effluents  tested.   The
values  represent  the range of response for the No Sexual  Reproduction
effect  (> 957. decrease from the control).   The number  in  parenthesis
after the~"effluent type is the number of effluents of that type tested.
     EFFLUENT TYPE                       RANGE OF RESPONSE (7.)


     Heavy Metals (2)                         0.05-0.5

     Pulp Mill (1)                            1.8

     Organics (5)
      (pesticides, dyes, etc.)                3-20 (generally <5)

     Sewage (17)                              2.5-25 (generally 5-10)
                                   III-5

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Table  2L.   The  effect  of effluent from ITT Rayonier on  formation  of
cystocarps  by Champia parvula.   Temperature was 23.to 25 °C,   salinity
was  30°/oe,  and  light  density  was ca 100 uE  m"   3"   of   daylight
fluorescent  light on a 16h:8h,  light:dark  cycle.    The  concentration
resulting in no sexual reproduction (_>957. decrease from control) was 2.5
7.  effluent with both test runs.   The data from the May 15th  and   19th
test  runs  also had 2.5 7. as the cut off point even though  these   data
were considered unacceptable.  Mo necrotic tissue was observed  at any of
the concentrations tested.
       Effluent                  Number of Cystocarps per Plant (n-3)
                                       May 17          May 18
Control
0.5
1.0
2.5
5.0
10.0
11 ± 2
12 ± 3
13 ± 1
0.2
0
0
14 ± 5
13 ± 3
9 ± 3
0
0
0
                                     III-6

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Table 3.   The effect of receiving waters  from the Amelia River on  the
formation of cystocarps by Champia parvula.   Temperature,  salinity and
light  conditions were the same as for the  tests in Table  1.   Effluent
percents were calculated based on the dye study. No necrotic  tissue was
observed in any of the treatments.  A blank space means missing data.
Sta. No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
% Eff .
2.0
b

1.5

0.05
1.4
0.08
1.4
0.5
0.2
0.9
0.7
0.5
0.5

0.6
0.5
0.4
0.4
0.5
Number of Cystocarps per Plant
May 17 May 18
Replicate3 Replicate
ABC Mean A B
0.4 0.8 0.2 0.5* 0.0
17.2 12.2 14.6 14.7 20.6
1.0 1.6 0.4 1.0* 2.0
11.8
4.6 1.6 0.6 2.3* 0.0
1.6
2.4
1.6 11.6 11.6 8.3 23.6
1.2
14.4 15.4 15.8 15.2 10.4
10.8 13.2 14.4 12.8 26.6

8.4
2.0
5.8
6.0
12.0
7.8
15.0
10.8
9.2
1.6
17.4
4.0
9.0
1.2
0.6
4.4
18.4
1.0
16.6
13.3

6.0
8.6
4.6
9.8
11.4
11.6
5.8
9.6
10.0
Mean
0.8*
19.0
3.0*
10.4
0.6*
1.1*
3.4*
21.0
1.1*
13.5
19.9

7.2
5.3*
5.2*
7.9
11.7
9.7
10.4
10.2
9.6
a Mean of five plants per replicate.

  Control station, assume effluent 7. to be close to  zero.

* Statistically less than station 2 (ANOVA followed  by Dunnett's mean
  separation test).
                             III-7

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    25
    20
                                                     • Ambient
                                                     O Effluent Dilution
 6  I  5
5  I5o
Q.
cr
i  10
h-
Cfl
o
                •
             •Sto.4
                      \
                        \
                          \
                            \
                              X
           Sta.6
                 0.5
 1.0        1.5
%  EFFLUENT
                                                    2.0
2.5
Figure  1.    Number of cystocarps  plotted againsts 7. effluent  for  data
from  May  18th.   The  7.  effluent was based on the dye  study  of  the
previous week.   Plants treated with receiving water from station 4  had
an  unusually  large number of cystocarps for the  effluent  7..   Plants
treated  with  water  from  station 6 had an  unusually  low  number  of
cystocarps.  This  may  be due to the time difference between  the  dye
study  and   the toxicity  testing.   However,   considering  this  time
difference, the rest of the data show a good correlation.

-------
   20 r
                                         A Effluent  Dilution
                                         O NH4CI
    0 L
Figure  2.    Number  of cystocarps (as 7. of control) plotted  against  7.
effluent.    The effluent data are averages from May 17th and  18th.   The
ammonium chloride data are based on 70 mg NH,-N/L in the  effluent.   A
stock solution of 26.7 mg NH.C1/100 mL was used.

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                           PART IV
PRELIMINARY GUIDANCE MANUAL FOR CONDUCTING SEXUAL REPRODUCTION




    TESTS WITH THE MARINE MACROALGA,  LAMINARIA SACCHARINA,




             FOR USE IN TESTING COMPLEX EFFLUENTS
                             IV-1

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INTRODUCTION




     A  test  was  developed to use kelp,  Laminaria  saccharine,  as  a




toxicity test species for short-term exposure  to  toxicants.   A test has




also been developed using the red alga Champia parvula,  however,  this




alga  is  primarily a warm water species.   The kelps are more  normally




found  in colder waters,  and thus would make  a good complimentary  test




organism.  As with Champia, sexual reproduction was used as the endpoint



for accessing the effects of toxicants with Laminaria.  The test differs



from  that  of Champia in that it requires preconditioning in  order  to




perform tests.



     The  current Laminaria test is based on techniques that were  first




used  with  oil studies several years ago (Steele and  Hanisak,  1979).




Those  tests  used  material derived from nature  for  each  experiment,




hereas the present test arelies on cultured gametophytic material.
                                 IV-2

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MAIHTEHABCE OF STOOL COLTDBES OF T-AMTHARTA
     Laminar ia* 3   life   history  is  an  alternation  of   microscopic




gametophytes  with a. large diploid blade.   The clones  presently  being




used  were  isolated  from Rhode Island  in 1985.   It  is  probably  not




essential to have a standard clone, however, some experience is required




to  isolate new clones from the field  into unialgal  culture.   Unialgal




stock  cultures  are  necessary to maintain  healthy,  actively  growing



plants for use in testing.




     Male  and  female plants of Laminaria can be maintained  easily  in




unialgal  culture  in the laboratory.    New cultures can be  started  by



blending  old cultures,  and splitting into several new culture vessels.




Blending is accomplished using a food  blender at its fastest speed  (for




approximately  1  min).   For maintenance cultures,  a  nutrient  medium




without  added iron is used to inhibit gametogenesis;  allowing  greater




vegetative growth.  Some preconditioning is required to induce reproduc-




tion.  Under  the conditons listed below,  male and female  gametophytes




will produce gametes.  Thus, plant material can be available at any time




for testing.



     Unialgal stock cultures of both males and females are maintained in




separate,  aerated  500  mL Erlenmeyer flasks containing 400 mL  of  the



culture medium.   The choice of these  flasks is one of preference rather




than necessity.  The maintenance culture medium is artificial or natural



seawater to which additional nutrients are added (Table 1).  Seawater is



autoclaved  for  30 min at 15 psi.  The culture flasks are capped  with



aluminum foil and autoclaved dry, for  10 min.  Culture medium is made up



by  dispensing  seawater into the sterile flasks and adding  the  appro-



priate  nutrients from a sterile stock solution.   Alternately,  500  mL
                               IV-3

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flasks could be autoelaved with the seawater already in them.   Sterili-



zation  is  used to prevent microalgal contamination,  and not  to  keep



cultures bacteria-free.



     We  recommend  that several cultures of both males and  females  be



maintained  simultaneously to keep a constant supply of  plant  material



available.   Initial stock cultures should be started weekly or biweekly



with  freshly  blended material.   Cultures are gently  aerated  through



sterile,  cotton-plugged, disposable, polystyrene 1 mL pipets.  Cultures



are  capped with foam plugs and aluminum foil and illuminated with ca 75


    -2  -1
uE m   s   of cool-white fluorescent light on a 16:8,  light:dark cycle.



The temperature is 12 to 15 °C and the salinity 28 to 30°/ao.  Media are



changed   biweekly.   About one-half to one-third of the plant  material



should be placed into another culture vessel with each medium change  to



reduce the amount of biomass as the plants grow.   With the above proce-



dure,  actively growing  plants will be continuously available. The total



number  of cultures^maintained will depend on the expected frequency  of



testing.   We keep 7 actively growing cultures of each sex.  In this way



we  can use a different  culture each day and not reuse it for at least a



week; allowing ample time for regrowth of the blended material.





ARTIFICIAL
     Because  salinity  adjustments will be necessary  in  testing  most



complex  effluents (particularly their receiving waters),  an artificial



seawater recipe that yields good growth of Laminaria is  desirable.   An



artificial  seawater would also make  the test method more readily avail-



able to laboratories that do not have access to clean natural  seawater.



The recipe for the artificial seawater that we use is listed in Table 2.



Comparisons  between  the sensitivity to effluents in this  medium  with
                                       IV-4

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that in natural seawater have not  yet been conducted.






PREPARATION OF PLABTS FOR A TEST




     Stock  cultures  should  be checked for their readiness for  use  in




toxicity  tests.   Plants can be checked by examination under a compound




microscope  (50-100X).   Healthy gametophytes of both sexes  are  highly




branched and the condition of individual cells can be accessed by obser-




ving  the  chroma tophores.   These should appear as discrete pale  brown




discoid  objects  evenly dispersed in the cell.   Senescent  cells  will



appear  much darker and the chroma tophores will not  be  discrete.  Once




cultures are determined to be usable  for toxicity testing, plants should




be  blended using a commercial food blender at the highest  speed.   The




resulting  suspension  should  be  filtered through  a  30-60  urn  nylon




screening,  and the portion that passes through used for testing.  These




cells can be diluted and pipetted into the test chambers.
     Host testing to date has been performed in ca. 30 mL (using 60x25mm




petri dishes).  These dishes offer the advantage of being disposable, as



well  as  being  deeper than the standard petri dish  (Lab  Tek  #4036).




Recovery chambers can 'be either these dishes or scintillation vials.






TEST CUHDITOBS



     Temperature, Salinity,  Light and Aeration



     The test exposure duration is 2 days followed by a 3-7 day recovery



period  for females  for the development of sporophytes.   The  exposure



temperature  should be between 10-12°C,  and the salinity should be  be-




tween 28-32°/oe*  For receiving waters, salinity will often be below the



desired  range  and must be  adjusted with  artificial  sea  salts.   The
                                     IV-5

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                                                              •2  -1
photoperiod should be a 16h:8h,  light:dark cycle of ca. 75 uE m   s   of



cool-white  fluorescent  light.    Plants  are recovered under  the  same



salinity and light conditions,   but  the temperature is raised to 16°C to



slow  the growth of the females  while the sporophytes develop  (this  is



not essential, but it makes the  counting of the sporophytes easier).



     Nutrient



     The  nutrient recipe is listed  in Table 1.   Trace metals and  EDTA



are  omitted from the medium during  test exposures (except in artificial



seawater, where EDTA is added at 20  ug/L).





FB0TOGOL



1.   At least one week prior to  testing,  blend males,  filter and dilute



and  allow to settle onto cover  slips (100-200/cover slip).   The  minus



iron  medium  is used at this point.   This allows the males  to  attach



better to the cover slip before  gametogenesis begins.



2.   After  2-3  days replace medium for males with  one  that  contains



double strength iron/EDTA.



3.   On  the same day as #2, blend  the females,  filter and dilute  and



allow  to settle onto cover slips or small pieces of glass slides  (100-



200/slide). The complete medium  containing double strength iron is used.



4.   After  an  additional  4-5  days check males  and  females  for  the



presence of gametes.  If gametes are seen then testing can begin.



5.  Set  up and label control and treatment dishes;  three per treatment



and controls.



6.   Fill  dishes with 30 mL of  control or treatment water.  Use the 1/8



strength  nutrient  medium minus trace elements and  with  the  adjusted



EDTA.



7.   Add  one  cover slip (or glass  slide) each of  males  and  females.
                                      IV-6

-------
Rinse males briefly in seawater to remove loosely attached plants.  This

will minimize the transfer of males to  the slide containing the females.

8.   After two days,  remove slides containing females, rinse briefly in

seawater  and place into control medium (complete nutrients).   Transfer

to 16-18'C if possible.

9.   After  an  additional  3-7 days examine females  under  a  compound

microscope and count the number of sporophytes.

10.   Data  are  analyzed by analysis of variance  (ANOVA)  followed  by

Dunnett's  mean  separation  test  to  determine  differences  from  the

control.



NOTE:  We recommend making up four nutrient solutions.

     1. Complete minus iron; for maintaining stock cultures.

     2. complete plus iron; for recovery medium after exposure.

     3. 1/8  strength minus trace elements and EDTA;  for the  exposure
        medium.

     4. an  iron/EDTA  solution for increasing the  iron  concentration
        during gametogenesis.
                                       IV-7

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Table  1.   Recipe  of nutrients to be added to  artificial  or  natural
seawater for stock cultures and test medium.  The test medium concentra-
tions  are  1/8 strength of the stock culture concentration  except  for
EDTA.   The  concentrated stock solution  is autoclaved at standard  tem-
perature  and  pressure for 15 min (the pH is adjusted to 2.0  with  HC1
prior to autoclaving to prevent precipitation).
COMPOUND
NaN03
NftH , PQ . *E*—O
2 u 2
Na2EDTA.2H20
NaC,H-0, (citrate)
O J 1
Iron0
Trace Elements
Na2Mo04.2H20
KI
ZnS04.7H20
NaV03
MnS04-H20
Vitamins
Thiamine*HCl
Bio tin
B12
AMOUNT/LITER
STOCK CULTURES CONCENTRATED STOCK*
127 mg
12.8 mg
2.66 mgb
1.03 mg
195 ug

24.2 ug
83 ug
21.8 ug
6,l_ug
0.61 ug

1.95 mg
1.0 ug
1.0 ug
12.7 g
1.28 g
266 mg
103 mg
19.5 mg

2.42 mg
8.3 mg
2.18 mg
0.61 mg
61.0 ug


10 mLS

*Use  1.25  mL/L for test medium (with the adjusted EDTA  concentration)
 and 10 mL/L for stock cultures.   For test media an additional 2.5 mL/L
 of the sodium bicarbonate stock solution is added (see Table 3).

 The  concentration  is  20  ug/L for final solution of  test  media  in
 artificial seawater;  EDTA is omitted entirely  if this nutrient  medium
 is used in natural seawater.

CIron  stock  solution prepared by dissolving 1  g iron powder in  10  mL
 concentrated  HC1  and  diluting  to  1  liter  with  deionized  water.
 Acceptable  stock  solutions could also  be made with ferric or  ferrous
 chloride.  Iron is omitted for maintenance culture medium and is double
 for initiation of gametes.

 Trace elements are omitted for toxicity  test medium.


Vitamin  stock  solution autoclaved separately  in  10  mL  sub-samples.
 Each 10 mL contains 195 mg of thiamine.HCl,  100 ug biotin,  and 100 ug
 B12.  Adjust pH to ca 4.0 before autoclaving for 2 min.

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Table 3^. Recipe for artificial seawater using GP2. The concentrations of
the  salts have been adjusted to give a final salinity of  30°/00.   The
original  recipe  calls  for autoclaving anhydrous and   hydrated  salts
separately to avoid precipitation.   However,  if the sodium bicarbonate
is autoclaved separately (dry),  then all of the salts can be autoclaved
together.   Since no nutrients are added until needed,    autoclaving is
not critical for effluent testing.  To minimize microalgal contamination
the  artificial seawater should be autoclaved when used for  stock  cul-
tures.   Autoclaving  should be for at least 10 min for 1 liter  batches
and  20  min  for 10 to 20 liter volumes (at  standard  temperature  and
pressure).


        COMPOUND                        GRAMS/LITER*
MaCl
Na2S04
KC1
KBr
N.2B407-10H20
MgCl2.6H20
CaCl2.2M20
SrCl2-6H20
HaHC03b
21.03
3.52
0.61
0.088
0.034
9.50
1.32
0.02
0.17
Generally made in 10 to 20L batches.

 A  stock  solution of 68 mg/mL sodium bicarbonate is prepared by  auto-
 claving it as a dry powder and then dissolving it in sterile  deionized
 water.  For each liter of GP2 use 2.5 mL of this stock solution.

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REFERENCES




Steele,  R.L.  and M.D. Hanisak.  1978.   Sensitivity of some brown algal




     reproductive  stages to oil pollution.    In:   Proceedings  of  the




     Ninth International Seaweed Symposium,   pp. 181-190.  A. Jensen and




     J.R. Stein (eds).  Science Press.

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                     PART V
COMPARISON OF THE SENSITIVITY OF LAMINARIA WITH




                THAT OF CHAMPIA
                      V-l

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INTRODUCTION




     At  this point,  Laminaria looks  very promising as a  toxicity  test




species for testing single compounds and complex effluents.   However, a




comparison  of test results using Laminaria with that of the test  using




Champia was necessary to determine the sensitivity of the new  procedure




to  toxicants.   The comparison was made using  two heavy metals  (silver




and  copper);  two organics (pentachlorophenol  and isophorone);  and one




sewage effluent (East Greenwich STP).






MATERIALS AND METHODS




     The  procedures  used for both Laminaria and Champia  are  describe




elsewhere in this report (Parts IV and I respectively).






RESULTS




     The results of the comparison of  Champia and Laminaria are shown in




Figures 1 and 2 and Table 1.  The sensitivity of Laminaria to copper and




silver  was  not  as great as that for Champia   (Figure  1).   In  fact,




Champia  was at least an order of magnitude more sensitive.   However,




Laminaria  and Champia were very similar in their sensitivities to  both




of  the  organics tested (Figure 2) and the sewage effluent  (Table  1).



     The results of these early comparisons indicate that Laminaria will




be  a useful toxicity test species.   It was noteworthy that  these  two



algal  species,  which  come  from  vastly different  phyla,  responded



similarly to the organics and sewage effluent.   The gives support to the



use  of  either  species to make preliminary generalizations  about  the




response of seaweeds to toxicants.
                                 V-2

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Table 1.   The effect of sewage effluent from  the East Greenwich STP  on
sexual  reproduction in Laminaria saccharins and Champia  parvula.   The
values  listed  are  the geometric mean (as  percent  effluent)  of  the
effect/no  effect  concentrations  using no sexual reproduction  as  the
effect measured.
          Date
Laminaria
Champia
    September 9, 1986

    September 15, 1986
   1.8

   3.7
  3.7

  1.8
                                  V-3

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     100
 o
 cr
 i-
 z
 o
 o
 u_
 o
 0.
 O
 (T
 O
 0.
      20 -
          ,1
                             40        60       80
                           O  COPPER  (^g/L)
                                                             100
                   10
            20        30

            '  SILVER
                                                  40
50
Figure  1.  The  effect of copper and silver on sexual reproduction  in
Laminaria  aaccharina and Champia parvula.   The effect for  Champia  is
           only
represented
occurred.
by an arrow where the total absence of  reproduction

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   100



J^
t
o 80
/g/L) 1
i i i i i t
0 200 400 600 800 1000
                           ISOPHORONE  (//M)
Figure  2.   The  effect of pentachloxophenol and isophorone  on  sexual
reproduction  in Laminaria saccharina and Champia parvula.   The  effect
for  Champia  ia  represented  only by an arrow  where  the  absence  of
reproduction occurred.

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                                   TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
1. REPORT NO.
                              2.
                                                            3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
                    Development  of short-term Exposure
     Tests for Marine Macroalgae for use in Effluent
     Testing
             5. REPORT DATE
                  November,  1986
             6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
           Glen B. Thursby
                                                            8. PERFORMING ORGANIZATION REPORT NO.
                        X117
9. PERFORMING ORGANIZATION NAME AND ADDRESS
     Botany Department
     University of Rhode Island
     Kingston, RI 02881
              10. PROGRAM ELEMENT NO.

               B101
              11. CONTRACT/GRANT NO.
                                                              CR8.12070-01
12. SPONSORING AGENCY NAME AND ADDRESS
   Environmental Research  Laboratory - Narragansett
     South Ferry Road
     Narragansett, RI  02882
              13. TYPE OF REPORT AND PERIOD COVERED
              Final  Nov.  1984 - Nov. 1986
              14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES „,,.      •,-,,,.,.
                     iechniques  developed during this  cooperative agreement were
  presented at three Effluent Testing Workshops sponsored  by ERLN in 1985 and 1986.
16. ABSTRACT
 A previous  sexual  reproduction toxicity  test with the marine  red macroalga,  Champia
 parvula,  lasted two weeks.  This report  covers a modification to this  procedure in
 which males  and females are exposed  together to a toxicant or effluent for only two
 days.  The  procedure was used successfully with single compounds and a variety of
 complex effluents.   A comparison of  toxicity test results between  the  two-week and
 the two-day  exposure procedures was  performed using single compounds.   The two pro-
 cedures compared favorably.  Initial  steps were also taken in the  development of a
 similar two-day exposure test with the brown alga, Laminaria  saccharine.   Laminaria
 represent another  phylum of algae than Champia, and has the additional advantage of"
 being both  economically and ecologically important.  For the  current study,  the
 feasibility  of  using Laminaria as a  routine toxicity test species  was  verified.  A
 comparison was  also made between the  sensitivity of both species.   The sensitivity
 of Laminaria to copper and silver was not  as great as that for Champia.   However,
 both species were  very similar in their  response to pentachlorophenol  and isophorone,
 as well as  a sewage effluent.
 7.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS  C.  COSATI Field/Group
    Champia, Laminarja effluents
18. DISTRIBUTION STATEMENT

      Release Unlimited
19. SECURITY CLASS (This Report')
                                                                          21. NO. OF PAGES
                                86
                                              20. SECURITY CLASS (This page)
                           22. PRICE
EPA Form 2220-1 (Rev. 4-77)   PREVIOUS EDITION is OBSOLETE

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