United States
                   Environmental Protection
                   Agency
Environmental Research
Laboratory
Duluth MN 55804
                   Research and Development
EPA/600/S3-86/001 Mar. 1986
v>EPA         Project Summary
                   Control  of  Cadmium
                   Carbonate  Precipitation
                   Interferences During the
                   Dialysis  of  Cadmium  in  High
                   Bicarbonate Alkalinity
                   Aquatic-Life  Bioassay Waters
                   John E. Poldoski
                     The precipitation of cadmium car-
                   bonate during the dialysis of cadmium
                   in a high bicarbonate alkalinity natural
                   water, was linked to a significant
                   source of error when determining
                   dialyzate cadmium concentrations. The
                   relative  standard deviation was  re-
                   duced by approximately four-fold when
                   this  precipitation was controlled by
                   adding a particular preparation of
                   humic acid to the dialysis bag filling
                   solution. Linear regression correlation
                   coefficients  for sample-by-sample
                   comparisons between resultant dialysis
                   values and corresponding free cad-
                   mium values, obtained by cadmium ion
                   selective electrode, were 0.90 or
                   greater for concentrations in the range
                   of 2^
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Experimental Section

Apparatus
   Experiments were conducted using
two different  molecular weight cut-off
(MWCO) dialysis bags(Spectrum Medical
Industries, SMI),  specifically the 1,000
MWCO (#132634) and 12,000-14,000
MWCO (#132700) sizes clamped with
SMI closures.
   The flow-through mini-diluters were
operated as part of an aquatic-life related
study in this laboratory and details of this
work, including functioning of the mini-
diluters are described elsewhere (5,6).
   Atomic absorption  spectrometric
(AAS) measurements of total cadmium
and other  elements were made with a
Perkin Elmer model 5000 atomic absorp-
tion spectrophotometer equipped  with
deuterium  arc background correction, a
model  HGA-500  graphite  furnace,  a
model AS-40 autosampler, a  model AS-
50 auto-sampler, and a model 56 strip
chart recorder.
   Free cadmium ion and pH measure-
ments were made with  an Orion model
801 pH meter equipped with a 500 ml
FEP teflon cell, an Orion model  9448A
cadmium ion selective electrode (ISE), an
Orion  double  junction  reference  elec-
trode (#900200), an Orion  model 605
electrode  switch,  and a Perkin Elmer
model 165 strip chart recorder.
   Calculations were performed  with a
Texas  Instruments  model  T159  pro-
grammable calculator equipped with the
statistics solid-state  software module.

Chemicals and Reagents
   Unless indicated otherwise, all chem-
icals were of  reagent grade quality or
better and deionized distilled water (Mil-
lipore Super Q) was used for preparing
solutions. The high calcium hardness-
high bicarbonate alkalinity natural water
(hardwater), used as the dilutant water
for aquatic-life bioassays, was prepared
by dissolving CaCOafrom limestone (5,7)
in  Lake Superior water  (softwater)
followed  by filtration.  It was  subse-
quently aerated with 0.45 /u filtered  air
until a constant pH of approximately 8.2
was  maintained.  Non-complexing  di-
alysis retentate solutions (labelled as A
or B) were prepared to contain 4 x 10"' M
borate buffer (pH 8) and either 4 x 10~4 M
(A)or2x10"3M(B)Ca(N03)2.

Quenching  Reagent Preparation
   The quenching reagent was prepared
by adding  1,000  mg  of  humic  acid
(Aldrich, H1675-2) to one liter of hard-
water and shaking the mixture for 1  hr.
Portions of this solution were then pres-
sure-filtered at 3  atm through  a pre-
cleaned Millipore  0.45 fi, 47 mm di-
ameter membrane filter. The 0-10 mL
fraction was discarded and the 10-50 mL
fraction was collected and saved. This
filtration  process was repeated  with  a
new filter until the entire 1  L volume was
processed. The  collected filtrate  was
diluted ten-fold with deionized distilled
water and stored in the absence of light at
4°C until use.

Results  And Discussion

Reproducibility at High and
Low Alkalinity
   Table 1 presents replication data for 5
h dialyses of cadmium and other cations
in the mini-diluter bioassay system, in
addition  to  cadmium dialysis from
retentate  solution  B. For these cases,
deionized distilled water was used as the
filling solution to compare the effect of
various retentate solutions on reproduci-
bility. Data are generally presented as the
mean and relative  standard deviation of
ratios of total metal concentration in the
dialyzate to total metal concentration in
the unfiltered solution.  In addition, for
just hardwater, the average ratio (and its
relative  standard deviation) of  total
                 cadmium concentration in the dialyzate
                 to total cadmium concentration  in the
                 corresponding  0.45 n filtrate is also
                 given. Further, the means of the ratios of
                 each filtered  concentration to each
                 corresponding total metal concentration
                 are given for comparison. As expected for
                 the softwaters, with little or no precipita-
                 tion occurring,  reproducible  ratios close
                 to 1 were obtained. These comparisons
                 show that the filtrate and dialyzate ratios
                 are  very similar, suggesting a general
                 equilibrium  of  cadmium and other dis-
                 solved components  between dialyzate
                 and retentate solutions. For hardwater,
                 ratios significantly less than 1 were due
                 to a contribution from  paniculate forms
                 in  the  bioassay waters. In a  typical
                 hardwater flow-through bioassay sys-
                 tem, Cd2+ concentrations of up to 1 mg/L
                 were added to  the  high bicarbonate
                 bearing bioassay waters. This resulted in
                 the  solubility product  of cadmium car-
                 bonate being greatly exceeded with re-
                 sultant precipitation  continually oc-
                 curring in the water column and sticking
                 onto the surfaces of the tanks. Obviously,
                 this process could also be occurring in-
                 side the dialysis bags, possibly giving rise
                 to a significant source of imprecision and
                 positive bias. These reasons are likely to
                 explain  the  unusually high average  R
 Table 1.  Reproducibility of 5 h Dialyses in Various Carbonate Alkalinity Waters Using Deionized
         Distilled Water as the Dialysis Bag Filling Solution
Retentate Water Type
Metal
                  Percent
                  Relative
                  Standard
                  Deviation
              Number
 Retentate Solution B.
  No carbonate
  alkalinity

 Hardwater, 212 mg/L
  carbonate, alkalinity
  bioassay water
 Softwater, 48 mg/L
  carbonate, alkalinity
  bioassay water
Cd



Cd




Cd



Ca


Mg


Na
                                     1.02
 0.366
10312)'

 0.878
(0.948)
                                    0.912
                                    (0.968)

                                    0.757
                                    (0.816)

                                    0.968
                                    (0.948)
 6.1



45.4

34.0


 4.4



 1.0


 1.1


 1.0
12



10

10


14
 3Mean ratio of (total [metal] in dialyzate) / (total [Metal] in retentate).
 bIndicates mean ratio of (total [Cd] in dialyzate) / (total [Cd] in 0.45 u filtrate).
 cParentheses indicate mean ratio of (total [metal]in 0.45 u filtrate) / (total[metal] in unfiltered
  solution).

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value of 1.19 given for hardwaterandthe
corresponding  high  relative  standard
deviation  (34%-45%),  as  presented in
Table 1. Subsequent details will further
describe this problem and how it might be
avoided.
Quenching Effect ofHumicAcid
  Chemical  kinetics studies (8) of C02+
added to  hardwater,  with  and without
added clay, indicated that the initial re-
action rates exhibited pseudo first-order
behavior with respect to loss of Cd2+ from
solution  and  that they  were  greatly
affected by a variety of other parameters,
but most  importantly,  humic acid pres-
ence (Figure 1). In the absence of humic
acid, observed pseudo first-order  rate
constants  (arbitrary units) ranged from
moderate to relatively  high values,
particularly at  high pH  and  high  clay
concentration.  However,  under similar
conditions with  50 mg/L  humic  acid
present, observed rate constants were at
near zero values over a range of pH condi-
tions. In softwater, with or without clay or
humic acid present,  loss of Cd2+ from
solution  as a function of time was not
observed.  These  specific  observations
demonstrated the problem and the likely
benefit of employing humic acid in the
dialyzate.
Effect of Variables on the
Conditional Concentration
Factor (F)
   The effect of  using  the quenching
reagent preparation as the dialysis bag
filling solution  was investigated  with
regard to equilibration time,  reproduci-
bility, and possible accuracy of dialysis.
Since this solution would  undoubtedly
bind cadmium ions,  there would  ob-
viously be a  natural tendency  for cad-
mium to  concentrate in the dialyzate.
Therefore, it was necessary to determine
this concentration factor and  some
factors  affecting  it.  This  information
could permit  calculation of the concen-
tration that would have normally dialyzed
in the absence of the concentration effect
of the quenching  reagent.
   It is appropriate to mention at this time
that the  experimental system studied
(flow-through and fixed volume) had two
noteworthy characteristics: 1) the effec-
tive retentate volume was infinitely large
relative to the dialyzate volume, and 2)
the humic acid in the quenching reagent
contributed insignificantly to the overall
ionic content of the dialyzate. Therefore,
the phenomena commonly referred to as
Donnan  membrane equilibria  become
insignificant in this case and should not
be an additional factor complicating the
interpretation of data. Definition of F  may
be attempted by first considering  reten-
tate media containing only  dissolved
cadmium species capable of equilibrating
with the dialyzate, with F given by the
following expression:

  p -total [Cd] in dialyzate = [Cd]d
     total [Cd] in retentate   [Cd]r
                                            Retentate solutions A or B fulfill these
                                         requirements and, therefore, the concen-
                                         tration of the species that are both inside
                                         and outside the membrane should be
                                         equal at equilibrium. For bioassay media,
                                         the concentration  of  other metals and
                                         ligands were considered negligibly low in
                                         concentration,  except for cadmium,
                                         calcium, and  bicarbonate. Calcium and
                                         bicarbonate concentrations were kept at
                                         a constant level, consequently concen-
                                         trations of cadmium,  pH, and  dialysis
                                         time were studied as  main parameters
                                         primarily affecting the value of F.
                                            Figure 2 shows the effect of dialysis
                                         time (1-20 h) on values of F for retentate
                                         solutions A and B, all normalized to each
                                         corresponding mean 5 h F value rep-
                                         resenting a set of conditions. Conditions
                                         varied  by using either 1,000 MWCO or
                                         12,000 MWCO  membranes  and total
                                         cadmium concentrations in the range of
                                         <10-200/ug/L. As a result,  very similar
                                         dialysis rates  and  equilibrium  values
                                         were obtained regardless of the  par-
                                         ticular membrane. It can be seen that the
                                         value of F stabilized after 4 h and changed
                                         about  10% or less in the 4-20 h range.
                                         Reproducibility was in  the range of 10%
                                         or less  for the plateau  region.
                                           As anticipated, experiments that were
                                         conducted in high bicarbonate alkalinity
                                         bioassay water produced similar results
                                         with  respect  to  dialysis rate and  re-
                                         producibility  (Figure  3).  Under  actual
 74,

 72

 10

  8

*6

  4

  2
      7.4 7.6  7.8 8.0 8.2 8.4 8.6 88 9.0
                  pH

 Figure 1.    Water quality variables affecting
            the pseudo first-order rate con-
            stant (arbitrary units) for loss of
            free cadmium from solution.
             A - hardwater + clay (90 NTU
            turbidity) + 10 mg/L Ccf*.  •  -
            hardwater  + 10  mg/L  Cd2*.
             • - hardwater + clay (90 NTU
            turbidity) + humic acid (50 mg/L)
            + 10 mg/L Cd2*.
                                            1.0
   0.8
 .% 06
 ~
   04
                                          
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   bioassay conditions, a plateau  was
   reached  in 4-5  h. In addition, the im-
   provement in reproducibility, that can be
   seen by comparing data in Figure 3 (error
   bars indicate standard deviation) to cor-
   responding data in Table 1, strongly in-
   dicates that  the high error associated
   with dialysis  in this type of water can be
   significantly  reduced by using an ap-
   propriate quenching reagent in the
   dialyzate.
      1.2
     0.4
     0.2
       0     123456
                 Dialysis Time (h)

   Figure 3.   Change in mean 4 h normalized
              conditional concentration factor
              as a function of dialysis time for
              high bicarbonate alkalinity flow-
              through bioassay waters.  X -
              data to  which other data  are
              normalized.  The number of data
              points averaged for the 1, 2, 3.
              5, and 6 hour periods were 7, 7,
              2, 4. and 3, respectively.
        Literature Cited

        1) Benes, P. Semicontinuous Monitoring
          of  Truly Dissolved Forms of Trace
          Elements in Streams Using Dialysis In
          Situ I. Principle and Conditions. Water
          Res. 1980, 14, 511-513.
        2) Rainville, D.P. and Weber, J.H. Com-
          plexing Capacity  of Soil Fulvic Acid
          for Cu2+, Cd2+, Mn2+, Ni2+ and Zn2+
          Measured by Dialysis Titration: A
          Model  Based on Soil  Fulvic  Acid
          Aggregation. Can. J. Chem. 1982,60,
          1-5.
        3) Truitt,  R.E.  and  Weber, J.H. Deter-
          mination of Complexing Capacity of
          Fulvic  Acid  for Copper  (II) and  Cad-
          mium  (II) by Dialysis Titration. Anal.
          Chem. 1981, 53,  337-342.
4) Truitt, R.E. and Weber, J.H. Copper(ll)-
   and Cadmium(ll)-Binding Abilities of
   Some New  Hampshire  Freshwaters
   Determined by Dialysis Titration.
   Environ.  Sci. Technol. 1981,  15,
   1204-1208.
5) Benoit, D.A.  U.S. Environmental Pro-
   tection Agency, Duluth, MN, Personal
   Communication, 1981-1983.
6) Benoit, D.A., Mattson, V.M., and
   Olson, D.M. A Continuous-Flow Mini-
   Diluter System  for  Toxicity Testing.
   Water Res. 1982, 16, 457-464.
7) Lemke, A.E.  A  Water  Hardener for
   Experimental Use. J.  Am.  Water
   Works Assoc. 7963, 61, 415-416.
8) Poldoski, J.E. U.S. Environmental Pro-
   tection Agency, Duluth,  MN. Unpub-
   lished work, 1981-1983.
           The EPA author, John E. Poldoski (also the EPA Project Officer, see below), is
             with Environmental Research Laboratory, Duluth, MN 55804.
           The complete report, entitled "Control of Cadmium  Carbonate Precipitation
             Inteferences During the Dialysis of Cadmium in High Bicarbonate Alkalinity
             Aquatic-Life Bioassay Waters," (Order No. PB 86-145 620/AS; Cost: $9.95,
             subject to change) will be available only from:
                  National Technical Information Service
                  5285 Port Royal Road
                  Springfield,  VA 22161
                  Telephone: 703-487-4650
           The EPA Project Officer can be contacted at:
                  Environmental Research Laboratory
                  U.S. Environmental Protection Agency
                  Duluth, MN 55804.
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300

EPA/600/S3-86/001

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