TABLE 3.

ACCUMULATION OF V4c-pcnBTM BY FISH AND SNAILS IN RECIRCULATING STATIC MODEL ECOSYSTEMS AS

INFLUENCED BY TIME AND TREATMENT

Exper iment? TreatmentS

Days after start of experiment?

Organism 1 3 8 W 15 20 25 32 35 36 AZ 56
! ! Fish# 1,340.4 0,610.4 3,840.2 0.24041 a)
10 10.540,2 6.70.8 10,9 12,642.44 1.5 0.3
" 10 Fish 0.240 0.240 3,540.2 2,841.9 1,740.6 2.0+0.7 0.240.2
100 1.240.1 1,140.1 18,543.8 14,9#1.0 16,443.9 8,242.5 9,646.0
"
co 4 1 Snail s## 2.34161 2,041.3 0
10 8,442.7 0
" 10 Snails 3,741.1 4,644.1 0.4 1,741.4 0,340.1
100 44,0711.5 14,0744 1.9 5,642.5 1,240.3

* Tissue concentration (ppm) based on total '4c analysis (PCNB plus metabolites).

T 4 desorption phase was started after day 32 (expt. 1) and day 42 (expt. 11) by placing organisms in untreated

water.

f experiment | and 11 were conducted two months apart.

5 Concentration (ppm) of PCNB adsorbed to 400 grams soil.

# Gambusia affinis.

## elisomi sp.

** standard deviation.


REFERENCES
Kearney, P. C., A. R. Isensee, and A. Kontson. 1977. Distribution and
Degradation of Dinitroaniline Herbicides in an Aquatic Ecosystem.
Pesticide Biochemistry and Physiology. 7: 242-249.

Mount, D. I., and W. A. Brungs. 1967. A Simplified Dosing Apparatus for Fish
Toxicology Studies. Water Research. 1: 21-29.

27


S.S Screen

2.0 mm Openings
Screen Spacer
an Gloss Gote

. | / (Glow foal
Sp Gloss Partition

5.5. Screen
Air In 038 mm Openings

End View
(Port

jon)

'Percolator’ Water Pump

Water Level

000

|

Gloss Partition Dephnids

000000:

Algae

00:

Snails

00001

200

Side View

Figure 1. Recirculating static model ecosystem. Tank

is 41 x 20 x 24 cm and holds 16 liters of
water.


TABLE 6. SIMAZINE WATER CONCENTRATIONS* IN AQUATIC MODEL ECOSYSTEMS

Day of test
Experiment Treatment?
1 3 7
Static 0.1 0.740 0.9+0 1.4+0.1
1.0 8.14+0.8 10.440.4 14.240.2
10.0 100.0+3.8 137 .34+5.5 160.5+17.8
Flowing 1.0 10.244.8 1,040.2 1,140.3
10.0 43.843.4¢ 5.7+0.6 7.240.8
100.0 5.745.4F — 55.141.0 56.3+1.8
* ppb.

t Concentration of simazine adsorbed to 400 gram soil (static, ppm) and
theoretical concentration introduced into water (flowing, ppb).

¥ Diluter malfunction.

14


FOREWORD

There is a growing recognition that techniques are needed which are
capable of measuring effects that are beyond the single species level of
organization--yet reliable and readily useable techniques are few. One is
faced with the need to choose the best system for measuring a desired effect,

with little hard data on which to base such a decision.

This report provides such data for two model ecosystems
same organisms, but differing principally to the extent that
flow-through system and the other was a recirculating static
Conclusions are presented on the relative advantages of each

containing the
one was a
system.

for testing

chemicals of a range of degradability and for simulating distinctly different

environmental situations.

J. David Yount
Deputy Director

Environmental Research Laboratory-Duluth

iii


0
Figure 4. Adult + Gambusia affinis in vertical swimming position.

25


TABLE 4, ACCUMULATION OF 14c-pene* BY DAPHNIDS AND ALGAE IN FLOWING MODEL ECOSYSTEMS AS
INFLUENCED BY TIME AND TREATMENT

Days after start of experiment?

Experiment? treatmentS Organism 1 3 7 " 15 20 25 30 32 42
1 1 Daphnids* 0.6 0,440.3 0.6 0
10 24140,38# 1,340.1 2,341.4 3.2410
100 - ~
" 1 Daphnids 0,640.8 0,240.3 2.3 0,640.1
10 0,970.6 0540.6 7,041.7 3,341.6
100 8,642.9 4,442.2 72.9F17.2
\ ' AlgaeTM* 0 0.4 0.2 0
EB 10 7.242,2 4,040.6 4,440.2 0.8
100 16.4422.9 82,5415.6 17,246.2 53.1413.2
" 1 Algae 0.1 0.7540.75 2,040.5 1.0 0.140
10 0.240.2 36047434 9,475.4 1,840.8 0.34041
100 2,340.5 142,74156.4 472.0608 164.5485.0 11,3486

* Tissue concentration (ppm) based on total !4c analysis (PCNB plus metabolites).

t 4 desorption phase was started after day 30 by turning off the dosing apparatus In the serial diluter.
F Experiment | and I! were conducted two months apart.

S theoretical water concentration of PCNB in ppb.

# Daphnia magna.

## standard deviation.

** dedogonium cardiacum.


Thin layer chromatographic analysis of fish and snail extracts indicated
that different breakdown processes were occurring in the two systems. In the
fish homogenates from the static and flowing systems, 30-70% and 8-162,
respectively, of the total 14¢ was hydroxysimazine. In the flowing
system, a major unidentified metabolite, which moved near the solvent front,
accounted for the rest of the !4c. The snails contained both the
unidentified metabolite and hydroxysimazine in the same proportion in both
systems.

Trifluralin

The concentration of trifluralin in water reflected its treatment rate for
both the static and flowing systems (Table 9). These values are based on
extraction analysis of the water (ethyl acetate:hexane, 70:30). The direct
count analysis of water on day 15 and 30, highest rates only, indicate that
much of the !4c¢ in the static system was not trifluralin, while most of
the 14c¢ in the flowing system was.

Significantly different bioaccumulation ratios (BR) were observed between
the static and the continuous dosing systems (Tables 10-13). Ratios were
generally much lower for the static systems and were similar to the values
obtained from a previous study on the distribution of dinitroaniline herbicides
in the aquatic environment (Kearney, et al. 1977). Bioaccumulation ratios of
several thousand were obtained with the continuous dosing system; values which
are sufficiently high to indicate a potential environmental risk. Differences
in the design of the two ecosystems and the degradation characteristics of
trifluralin largely explain the variation in results. In the static system,
trifluralin in solution would degrade within a few days, leaving more polar
products in solution. These degradation products would be accumulated less by
the organisms than the unaltered trifluralin. However, it should be pointed
out that the BR were based on total 46 in tissue and extracted water.
Therefore, in the static system, the actual ratios between trifluralin in water
and tissue may have been about the same as for the flowing system, but the
concentration of trifluralin in water and tissue would have been much lower and
changing with time. In the continuous dosing system, organisms were exposed to
a continuous supply of unaltered trifluralin, which would allow for maximum
accumulation to occur. The results support this concept since the highest BR
values for the static systems were obtained on day 1 and 3 then decreased with,
time, whereas for the continuous dosing systems, BR values increased with time.
The tlc data also supports this idea; the fish and snails from the static
system contained little trifluralin and several metabolites while the tissue
samples from the continuous dosing systems contained almost entirely
trifluralin.

The effects data also illustrated the differences in the two systems. No
noticeable effects were observed in the static systems, while in the continuous
dosing systems (at the high rates) fish were observed with yellow bellies,
unusual swimming behavior, (i.e. swimming upside down, laying upside down on
the bottom, jerking, general inability to maintain a proper orientation in the
water column) darkening of the tail pigmentation, and what appeared to be a

17


algae were very high and erratic for Experiment II, possibly due to the mats of
floating algae directly receiving treatment water. Any undissolved PCNB would
then be directly deposited on the algae, resulting in unusually high
"accumulation" rates.

Organisms in the static systems appeared to reach equilibrium concen-
trations faster than in the flowing systems. However, at similar water
concentrations, tissue content for daphnids, snails and fish were reasonably
close.

Thin layer chromatographic analysis of snails and fish revealed that
considerable metabolism of PCNB had occurred, but the number of metabolites and
amount of each was nearly the same for both the static and flowing systems.

Simazine

Lower soil application rates were used for the static system (0.1, 1.0 and
10.0 ppm) to avoid possible toxicity to the algae. In the flowing system,
simazine was dissolved in an ethanol:water mixture (1:13) and metered into the
mixing chamber with a peristaltic pump. Dosing rates of 0.1, 1.0 and 10.0 ppb
were used. Water contents shown in Table 6 reflect a dosing apparatus
malfunction on day one, which resulted in a high simazine input to 1.0 ppm
tanks.

The amount of simazine accumulated by the various organisms is presented
in Tables 7 and 8. For the flowing system (day one) all organisms contained
much more simazine at the 1.0 ppb rate than at the 10.0 ppb rate, reflecting
the dosing apparatus malfunction. The simazine in these organisms desorbed
rapidly as the water concentration declined to normal levels. The organisms in
the static systems generally reached an equilibrium tissue concentration by day
one and did not accumulate appreciable amounts of simazine after that day
although a slight increase in tissue level did occur.

The snail tissue levels do not appear to be significantly different
between the two systems, which may reflect the snail feeding habits and the
simazine present on the various substrates. The algae and daphnia show higher
tissue levels of simazine in the continuous dosing system than those found in
the recirculating static, possibly reflecting the larger amount of simazine
available during the thirty days. The concentration of simazine in fish
(static system) increased with water level and did not change much over time.
In the flowing system, however, tissue content in fish was very erratic, both
between treatment rates and with time. We know of no explanation for these
results.

Most of the organisms readily lost simazine when placed in untreated water
(static) or when the dosing apparatus was turned off (flowing). The rate of
simazine loss was somewhat slower in the flowing system probably due to
residual simazine desorbing from tank surfaces.

13


soil particles. The flowing ecosystem simulates an effluent source of
compounds such as industrial or manufacturing effluent, where the supply is
reasonably constant and directly introduced into a waterway.

Chronic toxicity and effects studies are generally better run in flowing
as compared to the static systems. The principal reason is that the identity
and concentration of the toxicant is more clearly defined and controlled in the
flowing system. This is primarily true for degradable compounds. The toxicant
concentration would remain constant with time in the flowing system but would
change rapidly in static. Static systems could be used for toxicity testing if
the concentration of the parent compound was frequently determined as well as
the identity and concentration of metabolites. However, it seems obvious that
such an approach would require far more effort and the results may not be as
reliable (due to the presence of metabolites) as the flowing system. On the
other hand, for less degradable compounds and acute toxicity tests, the static
system could function as well or better than the flowing system.

The cost and supply of radiolabeled compounds can also influence the
choice between the static or flowing systems. Use of radiolabeled compounds in
ecosystem research is indispensable if the identity and concentration of
degradation products is desired. In our studies the flowing systems used at
least 10 times more labeled material than did the static systems. Therefore,
if there is a limited supply of labeled compound for a study, the use of the
flowing system may be in question.

The daphnid population was found to be more stable in the static than in
the flowing system primarily because the food supply was better. An addition
of "green water" to the flowing tanks improved the daphnid population
considerably. Therefore, the addition of food to the flowing systems, possibly
through the continuous dosing system, would probably increase the daphnid
population to near static system levels.

26


TABLE 1. PCNB* WATER CONCENTRATIONS IN AQUATIC MODEL ECOSYSTEMS

Days after start of experiment

Experiment’ Treatment! 1 3 4 7 ll 15 18 25 30
Static I 1 5.7+4.4 0 2,841.5 5.6+2.7
10 11.141.8 15.7+1.0 19.541.3 18.242.1
Static II 10 2.4 2.2 11.0 lel 1.0
100 10.0 7.8 7.8 7.4 4.2
Flowing I 1 1.3+0.3 0.8+0.4 0.7+0.9 0.7+0.3 2.14+2.0
10 5845.0 5.1F1.1 2.540.5  5.040.2 4.20.6
100 42.14+6.1 566743.7 65.74+1.0 58.7+0.9 60.5+0.7
Flowing II 1 1.4 0.8 1.9 0.6 0.3
10 3.2 1.0 1.8 0.5 2.9
100 2.9 5.3 13.3 8.0 24.0

* ppb

+ Experiments I and II were conducted two months apart.

f Concentration of PCNB adsorbed to 400 grams soil (static, ppm) and theoretical concentration
introduced into water (flowing, ppb).


TABLES
Number Page
1 PCNB water concentrations in aquatic model ecosystems ......++- 8

2 Accumulation of 14c-pcnB by algae and daphnids in recirculating
static model ecosystems as influenced by time and treatment... + 9

3 Accumulation of !4c~pcnB by fish and snails in recirculating
static model ecosystems as influenced by time and treatment... . 10

4 Accumulation of !4¢-pcnB by daphnids and algae in flowing model
ecosystems as influenced by time and treatment .« ...+-+ee« eee Ill

5 Accumulation of !4c-pcnB by snails and fish in flowing model
ecosystems as influenced by time and treatment ..+.-+-e+-++-s-+- 12

6 Simazine water concentrations in aquatic model ecosystems ....-.. 14

7 Accumulation of !4c-simazine by daphnids and algae in aquatic
model ecosystems as influenced by time and treatment . +... 15

8 Accumulation of !4-simazine by fish and snails in aquatic model
ecosystems ad influenced by time and treatment. . . 1... +++ + 16

9 Trifluralin water concentrations in aquatic model ecosystems. ... . 18

10 Accumulation of l4c-erifluralin by Daphnia magna in recirculating
static model ecosystems as influenced by time and treatment... . 19

Ll Accumulation of !4c-trifluralin by Oedogonium cardiacum in
model aquatic ecosystems as influenced by time and treatment... . 20

12 Accumulation of !4c-trifluralin by Helisoma sp. in aquatic
model ecosystems as influenced by time and treatment. ....... 21

13. Accumulation of 14¢-erifluralin by Gambusia affinis in aquatic
model ecosystems as influenced by time and treatment. ». ..... . 22

ix


broken back (Figures 3 and 4). The possibility that some of these effects may
be related to the presence of ethanol was not ruled out, however, previous
experiments in which ethanol was used at this concentration did not produce
these effects.

At the start of the experiment pregnant mosquito fish were placed in egg
laying chambers within the continuous dosing test chambers. This was an
attempt to determine the feasibility of using mosquito fish to obtain
information on biological effects in this system. At day thirty the adults
were harvested and the fry left in their respective chambers with untreated
water passing through. Sixty days later, after the fry had grown to near
maturity, they were harvested and analyzed.

Fry were born in all tanks except one control and one of the 100 ppb
tanks. The average trifluralin content of these fish was 0.13, 0.27, and 5.03
ppm for the 1.0, 10.0 and 100.0 ppb treatment rates, respectively. Analysis of
the 100 ppb treatment fish by tle indicated that 80% of the 14 was
trifluralin. These results indicate that trifluralin is slowly metabolized
and/or lost from fish.

The testing of three pesticides in the two model ecosystems has
demonstrated their relative utility, strengths, and weaknesses. We found that
the two systems cannot be routinely substituted for each other since
differences in their basic design can result in very different results for the
same compound (trifluralin in this study). The degradability of the test
compound must be considered in choosing between these two designs. Compounds
that degrade slowly and are therefore persistent in the environment would
likely behave similarly (accumulation rates, total amounts accumulated and
effects on organisms) in the two model ecosystems (PCNB and simazine in this
study). On the other hand, compounds that degrade faster (half life 2 weeks to
2 months) would likely behave differently between the two systems. Degradation
products would continuously be swept out of the flowing system so organisms
would be exposed mostly to the parent compound. In the static systems, the
amount of parent compound present in water would decrease with time as the
amount and number of degradation products increased. Thus, with degradable
pesticides, aquatic organisms would be exposed to a changing mixture of
compounds in the static system and primarily to the parent compound in the
flowing system. The response of the organisms would then depend on the
relative effect of the parent compounds vs the metabolites on the organisms.
For the static system, the theoretical ratio between the concentration of
parent compound in water and the concentrate in organism, at any one time,
should be about the same as the ratio obtained in the flowing system. However,
the determination of that ratio in the static system is far more difficult due
to the changing concentrations and presence of other degradation products.

Another major factor dictating the choice of systems is the likely mode of
introduction into the environment. The static ecosystem simulates an erosional
or sediment source of compounds, such as when a rainfall event causes runoff
from an agricultural area recently treated with pesticides. Under these
conditions pesticides would be transported into water adsorbed to suspended

23


0z

TABLE li.

ACCUMULATION OF VM4c—TRIFLURALIN® BY OEDOGONIUM CARDIACUM
IN MODEL AQUATIC ECOSYSTEMS AS INFLUENCED BY TIME AND TREATMENT

ExperIment Treatmentt

Days after start of experiment

1 3 7 15 30 35F 50
Static i] 0,1740,27§ 0, 1540.18 -- 0.15+0.18 0.0340,06 0,2440.13  0,14#0.22
(7758 (589) (75) (190)
10 1,65+1.09 0,56#0.17 0.70+0.40 —1,83+1.03 2.1240,80 1,2440.91  0,9040.36
(377) 70) (281) (278) (247)
100 38.05+15.12 5,050.93 15.23+2,87 31,5646,06 —-35,3844,11 25.43+7.87 — 16.6445.45
(791) (138) (276) (270) (147)
Flowing 1 0,0440.08 0,901.45 3,98+8.44 —1,26+1.28 1,46+1,30 0,5540.42 — 12,50#22.75
(250) (5294) (34811) (4480) (1929) ~ ~
10 0.140012 1,3841,71 33.12422.85 11,0746,92 3.1142.13  2,7040.76 — 38.43465.25
(474) (1467) (24678) (5760) (1983) 7 7
100 2.614055 41,4745,23 90.79432.42 112.30488.33 102.15+78.76 26,65+12.17  7,8243.18

(196) (2553) (5062)

(3858)

(3961)

* Tissue concentration (ppm) based on total '4c analysis (trifluralin plus metabolites).

t Concentration of trifluralin adsorbed to 400 grams soll (static, ppm) and theoretical concentration Introduced
Into water (flowing, ppb).

¥ atter day 30 the organisms were placed in untreated water.

5 standard deviation.

# Bioaccumulation ratio = tissue concentration/water concentration.


and the type of toxicity data desired. However, the flowing system is more
versatile in the types of data that can be obtained than either the static or
simple flowing chronic testing systems, but requires a higher level of design,
maintenance, and analytical input than the simpler systems.


ABSTRACT

This research project was initiated with the overall objective of
developing additional and better techniques for studying pesticides in aquatic
model ecosystems.

To achieve this objective, a model ecosystem was designed and built that
utilizes the continuous dosing, flow-through system routinely used for chronic
fish toxicity testing in combination with the organisms used in static model
ecosystem testing. A previously developed recirculating static model ecosystem
(simulating a sediment or erosional pesticide source) was simultaneously used
with the flowing water system (simulating an effluent pesticide source) to test
the behavior of three pesticides (pentachloronitrobenzene (PCNB), simazine, and
trifluralin).

Conditions in the static system favored pesticide degradation while the
flowing system insured continuous pesticide exposure to the organisms.
Pesticides were introduced in the systems at rates ranging from 0.1 to 100 ppm
(static; adsorbed to soil) and 0.1 to 100 ppb (flowing; directly into water).
Organisms included daphnids (Daphnia magna), algae (Oedogonium cardiacum),
snails (Helisoma sp.), and mosquito fish (Gambusia affinis).

The total amount of !4¢-1abeled PCNB (parent compound plus
metabolites) accumulated by all organisms was about the same in each of the two
ecosystems (for similar treatment rates). Pentachloronitrobenzene content in
organisms reached equilibrium levels with water in 3 to 7 days and decreased 50
to 95% when placed in untreated water for 10 days. Variability between samples
was high, primarily at the lower treatment rates and for algae. Analytical
problems and precipatation difficulties in the flowing system were responsible
for this variability. Simazine accumulation (parent compound plus metabolites,
14¢-l1abeled) by snails and fish was similar between systems for similar
treatment rates, while algae and daphnids accumulated higher amounts of
simazine in the flowing systems than in the static. Also, more degradation
products were found in fish extracts from the static than in the flowing
systems. Trifluralin was extensively degraded in the static system with very
little accumulation of 14 by the organisms. Further, little of the
14¢ accumulated was trifluralin. Large amounts of trifluralin were
accumulated by all organisms in the flowing systems, most of it being the
parent compound. Also toxicity to algae and abnormal behavior responses of
fish to the highest trifluralin level were observed.

The basic design of the two systems is sufficiently different that they

cannot be routinely substituted for each other without first considering such
factors as test compound degradability, likely mode of introduction into water

iv


TABLE 2. ACCUMULATION OF 14c-penp* BY ALGAE AND DAPHNIDS IN RECIRCULATING STATIC MODEL ECOSYSTEMS AS

INFLUENCED BY TIME AND TREATMENT

experiment? Treatment

Days after start of experiment?

Organisms 1 3 7

it 10
100

Algae#
Algae 1,0 2.1 2.2
6.2

Daphnids##

Daphnids 6.8 3.8

wo
es

NN
bo
so
bo@

wo
oh

42 45 56
0.1
2.2
1.3 0.9
14,6 6.8
0
O.t
16.6

* Tissue concentration (ppm) based on total '4c analysis (PCNB plus metabolites).

t 4 desorption phase was started after day 32 (expt. 1) and day 42 (expt. 11) by placing organisms

7 Experiment | and || were conducted two months apart.

5 Concentration (ppm) of PCNB adsorbed to 400 grams soll.

# Qedogonium cardiacum.

## Daphnia magna.

in untreated water.


FIGURES
Number Page

1 Recirculating static model ecosystem. Tank is 41 x 20 x 24 cm and
holds 16 liters of water «ss eee eee ee eee eee v ene 4

2 Flowing water model ecosystem. Tank is 63 x 32 x 41 cm and holds 60
liters of water 6 6 6 ee ee ee ee ee eee ee ee ee eee OS

3 Adult 0 Gambusia affinis with bent spine. . . 1 6 ee ee ee ew ee 24

4 Adult 0 Gambusia affinis in vertical swimming position ....... 25

viii


TABLE 7. ACCUMULATION OF '4c~S1MAZINE BY DAPHNIDS AND ALGAE IN AQUATIC MODEL ECOSYSTEMS AS
INFLUENCED @Y TIME AND TREATMENT

Oays after start of experiment

Organism Experiment — Treatmont# 1 3 7 15 30 3st 42
Daphnids? — Static 0.1 3 0 ou 0.01 0
cor
1.0 0.01 0.01 0.03 0.02 0.02
a) «2
10.0 0.21 0,20 0.21 0,22 0.31
ay om
Flowing Out 0.19 0,19 ) 0.13 o.81
G2) o
1.0 0.35 0,09 0.14 0.17 1.09
(53) 19)
10.0 0.23 0.19 0.17 0.81 3.1
rc) re)
Atgaote Stetle Ont ° 0 0.01 0.01 0.0140,01 0 °
“6 @
1,0 0,0240,07 0,0440,02 0,0340,01 0,0640,02 0,140.01 0,0540.02 0,0540.02
a fe)
10.0 0, 1940.09 0,354.05 0,46+0.08 0,38+0,06 0,9140,12 0,4040,04 0,7540.39
o o
Flowing 0.1 ° 0 0,2040,19 0,0940.08 0.0440.07 0,040.08 0
oD (82) ~ ~ ~
1.0 0.10 0,0240,03 0,1840.25 0,2240,06 0,2840.06 0,060.06 0,07
co) fa) ~ ~ ~
10.0 0.06 1,1540.39 1,7240,64 1,5440.44 1,2740,31 0,9240,46
a) ~ ~
* Concentration of simazine adsorbed to 400 grams soll (static, ppm) and theoretical concentration

Tntraducad Into. (#2 water (flowing, p90).
Tt atter day 30 the organisms were allowed to dasord In untreated water,
5S Tissue concentration (ppm) based on total Ip analysis (simazine plus metabotites),

# aloaccumulation ratios (In parenthesis) tissue concantratlon/water concentration,

# Cedogonium cardiacums

15


United States Environmental Research EPA-600/3-80-008
Environmental Protection Laboratory Januar y 1980
th

Agency Duluth MN 55804

Research and Development

GEPA Freshwater
Micro-Ecosystem
Development and
Testing of Substitute
Chemicals

IS



SECTION 2
CONCLUSIONS

The results of experiments where three pesticides were tested in two
aquatic model ecosystems have indicated that static and flowing-water models
are each valuable tools for determining the fate and behavior of pesticides
in the aquatic environment, but cannot be routinely substituted for each
other since differences in their basic design can result in very different
results for the same compound. The static model ecosystems (that simulate a
sediment or erosional pesticide source) used in these experiments provided
excellent conditions for chemical and biological degradation while the
flowing-water model ecosystem (that simulate an effluent pesticide source)
provided continuous chemical exposure to the organisms and suppressed
degradation. As a result trifluralin, which is subject to rapid degradation,
showed a high level of accumulation and biological activity in the flowing
system, but was rapidly degraded and not accumulated in the static system.
The two other pesticides tested, PCNB and simazine, were less subject to
degradation and as a result, far smaller differences between the systems were
observed. Research on the behavior of pesticides (or other chemicals) in the
aquatic environment may require use of more than one type of model ecosystem,
depending on information being sought and properties of the compound. This
study also showed that both the static and flowing systems can be used in
toxicity and effects studies. However, if the compound is reasonably
degradable, then the flowing svstem will give the most reliable information
for the least amount of effort. Toxicity information on less degradable
compounds can be obtained equally well from either system.

The cost and supply of radiolabeled compounds can be a determining
factor in choosing between systems since the flowing system uses 10 or more
times as much radiolabeled compound as the static.


SECTION 1
INTRODUCTION

It is essential that we obtain as much information as possible about the
fate and behavior of a chemical in and its effects on the aquatic environment
before substantive quantities are released into natural waters. The model
ecosystem, which can be built to simulate a desired part of the environment is
a useful tool in obtaining this information. At present, fate and behavior
studies are being carried out using static or single dose systems; flowing-
water systems are being employed to obtain effects and toxicological data. The
primary purpose of this project was to determine if characteristics of both
flowing water and static systems could be incorporated to produce more
comprehensive model ecosystems.


(za

TABLE 13. ACCUMULATION OF '4c-TRIFLURALIN® BY GAMBUSIA AFFINIS IN AQUATIC MODEL
ECOSYSTEMS AS INFLUENCED BY TIME AND TREATMENT

Days after start of experiment

Experlment Treatmentt

1 3 7 15 30 35 50

Static 1 0.1540,33§ 2.2142.23  0.2640.42  4,5744.48 —-241441,50 0.6940.74 —-01740.03
ciBay# (4495) (647) (6191) (2583)

10 1,1240,88 2,680.84 = 1.0540.89 4,1645.27 2.674322 0.3940.25 —-0.5040.50
(340) (1042) (496) (493) (296)

100 25.3743.82 35.9545.34 19,1845,59 11,6341.49 10.7442,43  4,08+1.33 11,8341 .69
(686) (1167) (345) (78) (68)

Flowing 1 1,0541,04 0,5640,45 1,2241,62 0.914093 2,58#2.61  1.8541.37 0.7240.65
(8750) (3794) (5969) (1668) (3036)

10 1,O741,15 2.394191 7.994112 54341698 12.72415424  2,0741,00 —-1,0240.59
(3591) (2576) (3706) (2344) (6709)

100 11,10#1,64 30,9447.68 77,65+25.38 120.59423,96 82.29419.46 31,20+9.04 ---
(1271) (1897) (3974) (4069) (3919)

* Tissue concentration (ppm) based on total '4c analysis (trifluralin plus metabolites).

t Concentration of trifluralin adsorbed to 400 grams soll (static, ppm) and theoretical concentration Introduced
into water (flowing, ppb).

t After day 30 the organisms were placed in untreated water.
5 standard deviation.

# Bioaccumulation ratio = tissue concentration/water concentration.


1Z

TABLE 12, ACCUMULATION OF 146-TRIFLURAL IN” BY HEL!SAOMA sp. IN AQUATIC MODEL
ECOSYSTEMS AS INFLUENCED BY TIME AND TREATMENT

Experiment Treatmentt

Days after start of experiment

1 3 7 15 30 35 50
Static 1 0,2340,065 0,09+0.07 0 0.0140.02 —0,0340.04 0,06+0,05 0,01+40,04
(10674 (361) (0) ) (23)
10 0,4940.29 0,5340.45 0,0240.03 0.2740.21 0,124.13 0,0940,05 0,030.04
(149) (61) () 2) (73)
100 5.5742.14 4,4242,62 2,3940.83 3.4640.82 -3.7341.76 2.0940.96 0.944028
(155) (125) (43) (23) 9)
Flowing 1 0.1540.14 0,1440.27 0,2240.40 0,3340.26 = 041240429 0.284018 0.174008
(1250) (824) (1260) (1017) 9)
10 0.4340.22 0,0740.48 3.3045.29 = 4,.8545.34 = 15442.32 0.6340.33 —-063140,09
(1583) (752) (2285) (2718) (535) ~ ~
100 2.4542.40 17,99417,07 31,07421,72 32,52+16.66 18.744+12,37  4,89431.47  4,30+2.01
(260) (1087) (1494) (1085) (878) ~ ~

* Tissue concentration (ppm) based on total '4c analysis (trifluralin plus metabolites).

Tt Concentration of trifluralin adsorbed to 400 grams soll (static, ppm) and theoretical concentration introduced
into water (flowing, ppb).

t after day 30 the organisms were placed in untreated water.

5 standard deviation.

# Bioaccumulation ratio

tissue concentration/water concentration.


Foreword
Abstract

Figures
Tables

1.
2.
3.
4.
5.

Introduction

Conclusions
Materials and Methods
Experimental Procedures

Results and Discussion

References

CONTENTS

vii

» iii
» iv
evill
» ix
. 1
. 2
. 3
. 6
. 7
+ 26


TABLE 5. ACCUMULATION OF 14¢-pcnB* BY SNAILS AND FISH IN FLOWING MODEL ECOSYSTEMS AS
INFLUENCED BY TIME AND TREATMENT

Days after start of experiment?

Experiment? Treatment’ Organism 1 3 7 "1 15 20 25 30 32 42
\ \ Snall# 0.1 0,840.9
10 5.742 .4## 5,842.4
100 48..2429.7## 82.2422.3 79.2412.6
ul 1 Snail 2442.3 408 0.24041
10 2.42.3 0,940.5 13,4713,2
100 7.80.9 28.1710.0 101.942.7
! I Fish*® 0,2 0.9 1,340.9 0.4
& 10 1.64166 3,640.5  44240.9  3..5+0.6
100 63.1730.1 59,940.6 64,7412.8 98,2419,2
u 1 Fish 0,841.0 0,340.4 0,740.5 0,640.2 0,440.3 0,540.2
10 0,540.7 0,240.3 2,441.0 4,340.8 5.34160 4,142.1
100 5.641.8 2,741.7 26.6411.7 52.0#15.0 75,0¥12.2 88.4422.0

* Tissue concentration (ppm) based on total '4c¢ analysis (PCNB plus metabolites).

tA desorption phase was started after day 30 by turning off the dosing apparatus In the sertal diluter.
¥ experiment | and 11 were conducted two months apart.

5 theoretical water concentration of PCNB in ppb.

# Helisoma sp.

## standard deviation.

**Gambusla affinia.


TABLE 9. TRIFLURALIN WATER CONCENTRATIONS* IN AQUATIC
MODEL ECOSYSTEMS

Day of test
Experiment Treatment?
1 3 7 15 30
Static 1 0.23 0.70 0.40 0.83 0.86
10 3.40 2.50 2.87 8.37 9.10
100 36.93 31.33 55.10 148.807 160.10
(223.92) (337.77)
Flowing 1 0.12 0.17 0.20 0.45 0.83
10 0.47 0.91 1.40 2.58 2.48
100 9.27 16.23 19.60 29.83 21.62
(25.33) (21.73)
* ppb.

+ Concentration of trifluralin adsorbed to 400 grams soil (static,
ppm) and theoretical concentration introduced into water (flowing,
ppb).

¥ values in parenthesis are one ml direct counts others are 50 ml
extraction values.

18


DISCLAIMER

This report has been reviewed by the Environmental Research Laboratory-
Duluth, U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views and
policies of the U.S. Environmental Protection Agency, nor does mention of trade
names or commercial products constitute endorsement or recommendation for use.

ii


Top view

9 mesh screen

Gloss barrier

32 mesh screen

To drain or
Water level recirculating pump
Algae
Snails 32
Daphnids mesh

screen

{

Side view

Figure 2. Flowing water model ecosystem. Tank is
63 x 32 x 41 cm and holds 60 liters of
water.


SECTION 5
RESULTS AND DISCUSSION

PCNB

Two PCNB experiments were conducted; the second to improve techniques and
correct problems that developed in the first experiment. In the static
systems, PCNB was introduced at the rate of 1 and 10 ppm (Experiment I) which
resulted in very low 4c water levels. Treatment rates of 10 and 100 ppm
in Experiment II yielded much higher l4c¢ water levels in the water (Table
1). Numerous problems were encountered in the flowing systems, namely,
mechanical malfunction of the dilution apparatus and precipitation of PCNB on
glassware of the dosing system. PCNB precipitated from both carrier solutions
(ethanol:Triton X-100, 7:3, Experiment I and triethylene glycol in Experiment
II) when they contacted water, resulting in lower than anticipated water
concentrations.

The data for the recirculating static systems is presented in Tables 2 and
3. Equilibrium water concentrations were reached in one day and remained
nearly constant with time (Table 1). Similar soil treatment rates (10 ppm)
resulted (with one exception) in similar water contents between the two
experiments. Tissue contents of PCNB generally reflected treatment rate, while
variations in tissue content with time continued to be high in both
experiments. These variations are higher than those obtained in some previous
studies in our laboratory using similar static systems with pesticides of
similar physical-chemical properties.

The data for the flowing systems is presented in Tables 4 and 5. Water
content of PCNB was much lower at the 100 ppb treatment rate of Experiment II
than for Experiment I, which was undoubtedly due to the mixing chamber
insolubility problems. Variations in water concentrations with time were also
more variable in Experiment II. The concentration of PCNB in the four species
of organisms is shown in Tables 4 and 5. Unfortunately, few sampling times
coincided (samples were taken approximately 10 days apart for Experiment I and
on days 1, 3, 7, 15, and 30 for Experiment II) so direct comparisons are
difficult. However, a general trend of similar concentrations between the two
experiments (for comparative treatments) is evident for daphnids, snails, and
fish. This similarity was unexpected considering that water concentrations
were different (particularly at the 100 ppb treatment level). The 100 ppb
treatment rate of Experiment II was not toxic to daphnids, as compared to
Experiment I, where the highest concentration prevented the establishment of a
stable population. Thus, the water concentration established by chemical
analysis was confirmed by biological response. The PCNB concentrations in


EPA-600/3-80-008
January 1980

FRESHWATER MICRO-ECOSYSTEM DEVELOPMENT AND TESTING
OF SUBSTITUTE CHEMICALS

by

Allan R. Isensee
and
Ronald S. Yockim
Pesticide Degradation Laboratory
Beltsville, Maryland 20705

Contract No. EPA-IA6-05-5811

Project Officer

John G. Eaton
Environmental Research Laboratory
Duluth, Minnesota 55804

ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804


TECHNICAL REPORT DATA ;
(Please read Instructions on the reverse before completing)

7. REPORT NO. 2. [3. RECIPIENT'S ACCESSION NO.
EPA-600/3-80-008
4. TITLE AND SUBTITLE 5. REPORT DATE : ;
. * January 1980 issuing date
Freshwater Micro-Ecosystem Development and Testing l=: PERFORMING ORGANIZATION CODE

of Substitute Chemicals

Allan R. Isensee and Ronald S. Yockim

7 AUTHOR(S) ig. PERFORMING ORGANIZATION REPORT NO.

lg. PERFORMING ORGANIZATION NAME AND AODRESS 70. PROGRAM ELEMENT NO.

1BA601
Pesticide Degradation Laboratory

11, CONTRACT/GRANT NO,
Beltsville, Maryland 20705

EPA-IA6-05-5811

12. SPONSORING AGENCY NAME AND ADDRESS 13. TYPE OF REPORT AND PERIOD COVERED

Environmental Research Laboratory ~ Duluth, MN

Office of Research and Development 14. SPONSORING AGENCY CODE
EPA/600/03

U.S. Environmental Protection Agency
Duluth, Minnesota 55804

75. SUPPLEMENTARY NOTES

76. ABSTRACT

This research project was initiated with the overall objective of developing additional and better techniques for
studying pesticides in aquatic model ecoayatens.

To achieve this objective, a model ecosystem was designed and built that utilizes the continuous dosing, flow-through
system routinely used for chronic fish toxicity testing in combination with the orgenisme used in etatic model ecosystea
testing. A previously developed recirculating static model ecosystem (simulating a sediment or erosional pesticide source)
waa simultaneously used with the flowing water system (simulating an effluent pesticide source) to test the behavior of three
pesticides (pentachloronitrobenzene (PCNB), simazine, and trifluralin).

Conditions in the static system favored pesticide degradation while the flowing system insured continuous pesticide
exposure to the organisms. Pesticides were introduced in the systems at rates ranging from 0-1 to 100 ppm (static; adsorbed
to soil) and 0.1 to 100 ppb (flowing; directly into water). Organisms included daphnids (Daphnia magna), algae (Oedogoniva
cardiacum), snails (Helisoma sp-), and mosquito fish (Ganbusia affinis).

The total amount of T4C-labeled PCNB (parent compound plus metabolites) accumulated by all organisms was about the
game in each of the two ecosysteas (for similar treatment rates). Pentachloronitrobenzene content ia organisms reached
equilibrium levels with water in 3 to 7 days and decreased 50 to 95% when placed in untreated water for 10 days. Variability
between samples was high, primarily at the lower treatment rates and for algae. Analytical problems and precipatation
difficulties in the Flowing system were responsible for this variability. Simazine accumulation (parent compound plus
metabolites, !4c-labeled) by snails and fish was similar between systems for similar treatment cates, while algae and
daphnids accumulated higher amounts of simazine in the flowing systems than in the static. Also, more degradation products
were found in fish extracts from the atatic than in the flowing systems. Trifluralin was extensively degraded in the static
system with very little accumulation of !4c by the organisms. Further, little of the !c accumulated was
trifluralin. Large smounts of trifluralin were accumulated by all organisms in the flowing systems, most of it being the
parent compound. Also toxicity to algae and abnormal behavior responses of fish to the highest trifluralin level were
observed.

The basic design of the two systems is sufficiently different that they cannot be routinely substituted for each other
vichout Eirst considering such factors as test compound degradability, Likely mode of introduction into water and the type of
toxicity data desired. However, the flowing system is more versatile in the types of data that can be obtained than either
the static or simple flowing chronic testing systems, but requires a higher level of design, maintenance, and analytical
input than the simpler systems.

KEY WORDS AND DOCUMENT ANALYSIS

DESCRIPTORS b. IDENTIFIERS/OPEN ENDED TERMS cosat Fcld/Group

Aquatic biology Bioconcentration 48 B
Toxicology Bioaccumulation 57 HPY
Pesticides Model ecosystem 68 DEG
Fish Microcosm 73D
Invertebrates
Biogradability
78. DISTRIBUTION STATEMENT 19, SECURITY CLASS (This Report] 21. NO. OF PAGES

Unclassified

20. SECURITY CLASS (This page] 22. PRICE

Release to public Unclassified
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION 15 OBSOLETE 115 COVEN PRIMING OFFICE 1960 -657-146.75849

28


TABLE 8, ACCUMULATION OF '4c-SIMAZINETM BY FISH AND SNAILS IN AQUATIC MODEL ECOSYSTEMS AS

INFLUENCED BY TIME AND TREATMENT

Days after start of experiment

Organism Experiment Treatment? 1 3 7 15 30 3st 42
Fishs Statle 0. 0.0140.02F 0,0440.04 0,0640.06 0,1240.06 0.1140,07 0,030.03 0
_ (Oe (aR)
1.0 0,2540.08 0,2040.03 0.4540.13 1,2240.75 0,3640,04 0,0940,01 0.05
~ 19) 2)
10.0 2,9841,69 2.9440.73 2.6140.05 4.6140.88 4.5140.84 2,010.82 0.09
Flowing 0.1 2,0740.69 2.7145.70 1.6442.76 0,0340.05 0.0140,02 0,0440.05 0,01
~ 435) 477)
1.0 2.7440.94 11,5086.25 2.5843.76 004714041 0,8641.46
7 (6765) 354)
10.0 22141466 0.2340,22 O.68tIA11 12141419 1.6042.41 0,2740.07 0,02
Snalls#* Static oO. 0 0 ° 0 ° 0 °
Co) 0)
1,0 0.0140.01 0.0140 0,0140.01 0,0240.02 0.0240 0.01 0
= O (ars) =~ ~
10.0 0.0640.01 0,1340.03 0.22#0.05 0.2140.03 0.2340.04 0.09 0.04
~ m @ ~ ~
Flowlag 0.1 0.6540.92 0,0240,02 0.0140.02 0,0540.05 0.0140.01 0.06 0.01
o @
1.0 1,2141.67 0.1140,16 0.1740,08 0,0140,02 0.06+0,07 0.03 0,07
(65) a ~
10.0 0,400.62 0.0440.04 0,28+0,07 0.2340,09 0,1640.06 0413 0.07
o @

“Tissue concentration (ppm) based on total '4c anatysis (simazine plus metabolites).

t concentration of simazine adsorbed to 400 gram soll (static, ppm) and theoretical concentration
Introduced Into water (flowing, ppb).

t atter day 30 the organisms were placed In untreated water.

5 Gombusia attinis.

# Standard deviation,

#8 Bioaccumulation ratio = tissue concentratlon/water concentrat lon.

Hellsoma sp.

16


RESEARCH REPORTING SERIES

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

Environmental Health Effects Research

Environmental Protection Technology

Ecological Research

Environmental Monitoring

Socioeconomic Environmental Studies

Scientific and Technical Assessment Reports (STAR)
Interagency Energy-Environment Research and Development
“Special” Reports

Miscellaneous Reports

This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.

@ONODARWN >

o

This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161


a
Figure 3, Adult 0 Gambusia affinis with bent spine.

24


TABLE 10. ACCUMULATION OF !4c-TRIFLURALIN® BY DAPHNIA MAGNA IN
RECIRCULATING STATIC MODEL ECOSYSTEMS AS INFLUENCED BY TIME AND TREATMENT

Days after start of experiment

Treatment
1 3 7 15 30

1 0.15+0.06T 0.05+0.07 0.49+0.25 0 0.08+0.11

(617)8 (84) (1293) (0) (69)
10 1.9+0.27 2.694+0.18 0.84+0.11 0.22+0.11 0.43+0.0

(576) (1107) (300) (27) (47)

100 19.55+0.80 19.644+1.9 13.99+2.44 6.40+0.93 5.19+0.20
(536) (628) (254) (43) (33)

Tissue concentration (ppm) based on total lag analysis (trifluralin
plus metabolites).

t+ Concentration of trifluralin (ppm) adsorbed to 400 grams soil.

# Standard deviation.

§ Bioaccumulation ratio = tissue concentration/water concentration
in parenthesis).

19


SECTION 3
MATERIALS AND METHODS

Ecosystems

Two types of systems were designed and built for this study. The
recirculating static ecosystem is a modification of static systems previously
used in this laboratory (Figure 1). The system as shown has a total volume of
16 liters, with the small compartment occupying about one-fourth of the tank
volume. The test chemical is introduced (adsorbed to soil) to the large
compartment and carefully flooded with water to avoid suspending the soil. A
water depth of 1 cm is maintained over the top of the glass partition. The
percolator water pump is designed to maintain the same pesticide concentration
on both sides of the glass partition and to transport fecal matter to the
larger chamber where daphnids may use it as food. The screen-gate combination
on top of the glass partition provides additional experimental flexibility to
the model ecosystem. With the gate in place, daphnids are protected from
predation which allows development of a stable population. With the gate
removed, daphnids are subject to predation as they move into the fish
compartment and thus complete the daphnid to fish food chain.

The flowing-water ecosystem (Figure 2) also includes a partitioned tank
with the same organisms as in the static, but receives the test chemicals in
the incoming water. The chemical is introduced in a proportional diluter,
similar to that described by Mount and Brungs (1967). Flow rate was
approximately 2 tank volumes (120 liter) per 24 hr. An activated charcoal
filter was built to remove !4¢-labeled compounds from the drainage water.

Organisms

The organisms used for all experiments were daphnids (Daphnia magna),
snails (Helisoma sp.), algae (Oedogonium cardiacum), and mosquito fish
(Gambusia affinis). An attempt to replace mosquito fish with flagfish
(Jordanella floridae) failed when we were unable to rear these fish. Flagfish
spawning and hatching of eggs were successful; however fry growth rate was far
slower than anticipated making their use in our ecosystems impractical.

Experimental Chemicals

The following pesticides were tested in the ecosystems; the fungicide,
pentachloronitrobenzene (PCNB), and herbicides, 2-chloro-4, 6-bis(ethylamino)-
S-triazine(simazine) and a,a,a,-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine~
Ctrifluralin). All these pesticides were ring 14c-labeled, 43.24, 90.91,
and 45.25 yCi/mg, for PCNB, simazine, and trifluraline, respectively, and the
chemical purity exceeded 97%. The 1he-1abeled pesticides were mixed with
unlabeled pesticides of equal or greater purity for the actual treatments.

3


SECTION 4
EXPERIMENTAL PROCEDURES
Ecosystem Preparation

In the static systems the pesticides were adsorbed to 400 grams soil
(Matapeake silt loam, pH 5.3, organic matter content 1.5%; sand, silt, and clay
content of 38.4, 49.4, and 12.2%, respectively) at the rate of 1.0, 10.0, and
100.0 ppm for PCNB and trifluralin and 0.1, 1.0, and 10.0 ppm for simazine,
then flooded with 16 liters of water. The amount of pesticide in water over
flooded soil was found (through preliminary tests) to be about 1/1,000 of the
soil concentration. Therefore, these soil rates were selected to approximate
the water concentration in the flowing systems. One day after flooding 24
fish, 24 snails, about 1 gram algae and approximately 200 daphnids were added
to each tank. The serial diluter was turned on to fill the tanks of the
flowing system at least one week before the test chemical was introduced.
During this time, the organisms were added (same number as for the static
systems) and the diluter system was checked for proper operation. Metering of
the test chemical through the diluter was started the same day as the organisms
were introduced into the static tanks. The pesticides were introduced at the
rate of 1, 10, and 100 ppb for PCNB and trifluralin and 0.1, 1.0, and 10.0 ppb
for simazine.

Sampling and Analysis

The sampling times shown in Tables 1-13 represent a treatment phase
followed by a desorption phase. ‘The desorption phase was achieved by
transferring the remaining fish, snails, and algae to untreated water (static
systems) or by discontinuing the input of the treatment chemical in the diluter
(flowing system). Two fish, two snails, about 30 mg algae and 15 to 20 mg
daphnids were taken at each sampling time. The daphnid samples were analyzed
by standard liquid scintillation methods. Fish and snails were homogenized in
methanol and the !4c-content was determined by liquid scintillation
analysis. Algae samples were either combusted (PCNB experiments) or oxidized
in a Packard Tricarb Oxidizer (simazine and trifluralin experiments). The
1404 (from either source) was analyzed by scintillation methods. Water
samples were taken for direct liquid scintillation analysis (duplicate l-ml
samples) and/or extracted with organic solvent and then analyzed. Water
samples did not always coincide with tissue sampling for the PCNB and simazine
experiments, but did for the trifluralin experiments. Snail and fish
homogenates were spotted on silica gel TLC plates (20 x 20 cm GF-234, E. Merck,
Darmstadt) and developed with the following solvent systems: PCNB, n-heptane:
acetone:methanol (70:30:2 v/v/v); simazine, toluene:acetone (85:15 v/v); tri-
fluralin, ethyl acetate:cyclohexane (1:1 v/v). Each plate was autoradio-
graphed for 1 to 2 weeks with Kodak No. Screen medical x-ray film, NS-54T and
then the labeled spots were scraped and the l4¢ activity determined by
standard liquid scintillation methods.

6