Nondisinfected sewage effluent

Ly

Vent \

Cl, ges —>

<S0_ gos

Gam.- Amphipod (Gammerus)
F.H~ Fatheod Minnow

B

Oechtorinated Nondisinfected Nondisintected
Te Gom. To FH. To Gam. To FH.
dikter - dituter ethuter diluter

Figure 2. Chronic test apparatus used for volumetrically dividing,
chlorinating, and dechlorinating the sewage effluent.


Percentage hatch of fathead minnow eggs did not differ in the
dechlorinated, ozonated, and control waters (Table 9). Although egg
hatchability was the lowest in dechlorinated tank 100 A, the hatchability
in the duplicate chamber (100 B) was similar to that in all other test
chambers. Egg hatchability in the control waters was similar to that

in the dechlorinated and ozonated treatments.

30


9%

Table 7, MORTALITY OF SEVEN FISH AND SIX INVERTEBRATE SPECIES IN ACUTE TESTS OF YARIOUS CONCENTRATIONS OF
FOUR EFFLUENT TREATMENTS AFTER 7 DAYS' EXPOSURE

(expressed as percentage)

Wondisinfected | Dechlorinated | Ozonated | Chlorinated
Test species 100% | 75% rooz | 75x | 1ooz| 75x | sox] 37%) 25% | 12z| 6x
Hh
Brook trout go | 45 0 | o o | o {100 | 100 |100 | 75{ 0
Coho salmon o | o o | o 0 | 0 {100 | 100 |100 jroo | 35
Fathead minnow-Test 1 0 | o o}o o| 0 | - |} 100 {100 | 40} 0
Fathead minnow-Test 2 -|- -{- - | - [100 | 100 }100 [200 | 15
White sucker 4o | 0 o | o s | 0 |100 | 100 |100 | 10 | 10
Walleye 25 | 35 20 | 20 20 | 0 {100 | 100 |100 | 45] 0
Yellow perch 5 | 20 o| 5 s | o |100 }100 | 60 | o| 0
Largemouth bass olo ol o o{ 0 {100 }100 | so | of o
Invertebrates
Amphipod test 1 3 | 5 as* | 35 zo] 5 | - | 100 |100° | 80 | 35
Auphipod test 2 - {- - {- - | -  |100 }100 | 95 {30} 5
Stonefly a | - - |- 5s] 5 100 | es | 70 Jas] 5
Caddtefly so | 25 - |- w | s |95 | 75 | 85 | 40] 5
Grayftah® - - - |- - - so | 30 | 5 |20| 5
ag Operculate onaii” o | o of] o o} a w | o |i | ol o
Puluonatr snail - |- - |- -|- so | 20 |10 | o| o

*suryiyal of controls became less than 90% after 72 hr.
rests with crayfish and operculate snatle extended through 12 days and 14 days, resnectively.


The amphipod and daphnid results were statistically analyzed with
computation procedures found in Biomedical Computer Programs (Dixon, 1971).
The production of Daphnia (young per adult) was compared with a 3-way
analysis of variance. The variables were treatment method, concentration,
and test trial. Fathead minnow survival and the amphipod reproduction
index were examined with a 2-way analysis of variance. Because the 20

and 0.6 nominal percentage effluent concentrations were not uniformly

used in all four of the effluent test treatments, these two concentrations
were not analyzed with these procedures. Comparisons of differences

among the disinfected and nondisinfected effluents and differences within

an effluent group were made according to Dunnett's (1955) method.

RESULTS

Results of the routine chemical analyses of fathead minnow and amphipod
test waters are given in appendix Tables 1~2. Increasing amounts of
sewage in the test tanks progressively lowered the dissolved oxygen
concentrations and pH and increased the levels of total hardness,
alkalinity, and acidity. Mean dissolved oxygen concentrations in the
test waters were all above 50% saturation. An individual measurement
less than 30% saturation occurred only once during the fathead minnow
test and was found in the highest concentration of nondisinfected
effluent (20%).

The concentrations of total residual chlorine and sulfite maintained in

the respective chlorinated and dechlorinated effluent concentrations are
presented in appendix Tables 3-4. The duplicate chambers in each of the

five concentrations of chlorinated effluent had slightly different mean
concentrations of residual chlorine. However, the calculated standard

deviations were generally similar for each corresponding duplicate tank pair.
Concentrations of total residual sulfite maintained in the dechlorinated effluent

test chambers were more variable than the residual chlorine concentrations.

35


SECTION II

RECOMMENDATIONS

Concentrations of total residual chlorine as low as 10 ug/l. had a
detrimental effect on survival and reproduction of aquatic life.
Ozonation and chlorination followed by dechlorination are suggested as
disinfection alternatives for protecting aquatic life. Neutralization
of chlorinated effluents by dechlorination is needed to alleviate

adverse effects on aquatic life in the discharge areas. Additional
investigations are needed to more fully define long-term no-effect levels
for sulfite and other dechlorinating agents in receiving waters and

the potential formation of harmful oxidation products by ozonation.


Resources (1971) at two plants was 0.023 mg/1., which is lower than that
reported for brook trout in the present study (0.083 mg/1.). For additional
comparisons the reader is referred to the review by Brungs (1973) and
recent compilations by Becker and Thatcher (1973).

This study also showed that the chlorinated effluent had long-term effects
on survival and reproduction of fathead minnows, amphipods (Gammarus
pseudolimnaeus), and Daphnia magna. Chlorinated effluent concentrations
of 10% and 5% greatly reduced survival of fathead minnows and eliminated

daphnid survival. Chlorinated effluent concentrations of 2.5%
significantly affected amphipod reproduction, and no daphnids survived
at this concentration. The highest mean concentrations of total
residual chlorine having no effect on fathead minnows, amphipods
(Gammarus), and Daphnia magna were 14, 12, and 2-4 ug/1., respectively.

Daphnia magna was the most sensitive species during life-cycle tests

in the chlorinated effluent. Daphnid populations that survived to
adulthood reproduced successfully. Arthur and Eaton (1971) exposed
Gammarus pseudolimmaeus and fathead minnows to chlorinated (as chloramines)
lake water for 15 and 21 weeks, respectively. The effect concentration
for the amphipods was 3.4 ug/l. and 43 ug/l. for fathead minnows.

These authors also cited work by Biesinger that chloramine levels of
approximately 1 yg/1. were lethal to Daphnia magna in 3-5 days. The long-
term results from both of these studies are substantially in agreement.
From field surveys Tsai (1973) suggested that 50% and 25% reductions in
fish diversity could occur at total chlorine concentrations of 0.1 and
0.025 mg/1., respectively. Therefore, protection of aquatic life requires
the reduction of chlorine residuals to concentrations near the limit of
detection.

Dechlorination of the chlorinated effluent with sulfur dioxide provided
protection to the aquatic species tested. Short-term mortality in the

45


Table 12, SURVIVAL AND REPRODUCTION OF DAPHNIA MAGNA DURING 2 WEEKS'

CONTINUOUS EXPOSURE TO NONDISINFECTED AND DISINFECTED EFFLUENTS

Dechtortnaced Chlortnace
Young? ‘Total Percentage
ger’ [Survival after| mater seven
aisie_[Faaye/i2 Gaye] found | concenttstton [7 days
+
af w]e | orf am |r) 1 | i260 afo} | ©} ©
20 ry
afao] © | aso] a | wo] |] oe sjo} _| o | o
af}ao} oe | svfan | 9} 9] worjam | ao} » | oa] v2 afo]} | 0 |
0 5
a} ao} 9 | asa] a6 | 10 | 10 | use] us | 0 | 9 | 57] oo sfo]} _}| o | o
afao] 7 | ss} a0 | | 9 | a239| 1x7 | a0} 2 | some | a3 ato] —} © |
5 as
a} ao] 10 | soso] a | ¢| | ose] 10s | 10 | 6 | 522} 9 ato] _}| o | o
af oo] 9 | ase] an | wo} 7] ox! us | ao] 0 | a2 | oo afs | oafan | se
2s 12
a| 7{ 6 | se} o | so] 9] m2] o | 7] 6 | me] 52 ats | ef se | 7m
a] eo} e | vol se | o| | oss} ee | | | zor] as ato | s| a0 | a9
a2 0.6
a{ of 9 | sf a | wf] of ex! ws | o{ «| | uo afo | of m |»
af] oe | soo] m | ao] of] sos] os | wo] 9] os] o ala | oa] ses |
° °
cours) 3| s| 8 | roof or | ro] | soz| rm | ao] 7 | 46] 67] ccontzory a] 10 | a0] 903 | 97
‘Second caste
alo] | of a | of _] of of sf «| 2] 95 apo} -f oe]
2» rt)
a} oof @ | swf ow | of _| of o | sf v9} ula sfo} _| o | o
alr] 6 | 205] so} | 9} an] x | of s} me] a ato} _| o | o
10 5
wah af 2 [sf o | 2] 2] x6] sz f 6] 6] am] a s}o} _| o | o
af ef 5 | 250) a | ao] x} ms} mo | 7} 6] a6] ss afo| | o | o
5% 2s
sf 7} 5s | sul os | of 9] ml m | 9] 9} ae] as sfo} _| o| o
ala] oe | sof os | wo] | al ez | ao] ao} ser] sz spo} _] of o
as aa
2 9 o 637) 70 10 10 855 ' 7 9 6 14 35 a} Ss S| 382 3
afr] o | aca} a2 | ao} 0] sof on | of 8] us| ow ajo | 9] 26 | as
aa 0.6
al wo} a | auf 2 | of 0] os} se | 5] 0] ow] 8 a] 0 | | m3 | 79
a} 1] so | as} oe | sof 9] anf wo | of 6] aso} oe a} 20 | 10] 928 | 93
° °
soosrey stg} 4 | al» | wl of sol os | a0} ro} 2} 26} Gconero) n]_7 | 7] ore | 6

“wean rota) residual chlorine levels for the six test concentrations (in yg/1.) vere: Tear 17124, 50, 14, 4, 2, and sonnessurable respectivel
‘eat 2- 136, 38, 7, 2, nomeasurable, and nonmeasurable, respectively.

“roung par adult = (total nuber found minus adults alive at 12 days) divided by (adults alive at 12 days).
“For both teste the taitial number of daphaide per test chesber vas 10.

43


SECTION VIII

REFERENCES

American Public Health Association. 1971. Standard methods for the
examination of water and wastewater. 13th ed. Washington, D.C. 874 p.

Andrew, R. W. and G. E. Glass. 1974. Amperometric titration methods for
total residual chlorine, ozone, and sulfite. Draft Report, National Water
Quality Laboratory, Duluth.

Arthur, J. W. and J. G. Eaton. 1971. Chloramine toxicity to the amphipod
Gammarus pseudolimnaeus and the fathead minnow (Pimephales promelas).
J. Fish. Res. Bd. Canada 28:1841-1845.

Becker, C. D. and T. 0. Thatcher. 1973. Toxicity of power plant chemicals
to aquatic life. For U.S. Atomic Energy Commission by Battelle, Pacific
Northwest Laboratories, Richland, WA, WASH-1249, UC-11.

Benoit, R. F. and N. A. Matlin. 1966. Control of Saprolegnia on eggs of
rainbow trout (Salmo gairdneri) with ozone. Trans. Amer. Fish. Soc.
95:430-432.

Biesinger, K. E. and G. M. Christensen. 1972. Effects of various metals
on survival, growth, reproduction, and metabolism of Daphnia magna. J.
Fish. Res. Bd. Canada 29:1691-1700.

Brungs, W. A. 1973. Effects of residual chlorine on aquatic life. J.
Water Pollut. Control Fed. 45:2180-2193.

Brungs, W. A. and D. I. Mount. 1970. A water delivery system for small
fish-holding tanks. Trans. Amer. Fish. Soc. 99:799-802.

Collins, H. F. and D. G. Deaner. 1973. Sewage chlorination versus
toxicity - A dilemma? J. Environ. Eng. Div., ASCE 99:761-772.

Dixon, W. J. (editor). 1971. Biomedical computer programs. Univ.

California Publications in Automatic Computation, Univ. California
Press, Berkeley. 600 p.

49


Number of Surviving Amphipods after 4, 10, 16, and
20 Weeks’ Continuous Exposure to Nondisinfected and
Disinfected Sewage Effluent

Survival and Growth of Second Generation Amphipods

after 30 Days' Continuous Exposure to Nondisinfected
and Disinfected Sewage Effluent

53

60

61


Table 11.

SURVIVAL AND REPRODUCTION OF AMPHIPODS AFTER 20 WEEKS'

CONTINUOUS EXPOSURE TO NONDISINFECTED AND DISINFECTED EFFLUENTS

Nominal Total
Percentage Pinal Final number Young Births
Sewage male/female gravid young Total per per Reproductive®
soncentration ratio females | produced | births | female | female index:
Nondisinfected
A fab 4 431 15 107.8 3.8 4.8
20
B 6/9 3 682 a1] 75.8 1.6 1.9
A 416 2 734 v 321.3 2.8 3.2
10
B 718 5 585 2B 73.1 1.6 2.3
A 5/7 4 783 20 11.9 2.9 3.4
5
B 1/8 8 649 15 81.1 1.9 2.9
A Ws 3 883 23 176.6 4.6 5.2
2.5
B 5f5 5 399 Bo 79.8 2.6 3.6
A WW 6 393 4 56.1 2.0 2.9
1.2
B 4/7 6 117 6 16.7 0.9 Ly
A 3/7 2 56 3 8.0 0.4 0.7
0
(Control) B 14 2 19 1 4.8 0.25 0.8
Chlorinated®
A o/0 0 0 0 o 0 o
10
B 0/0 0 t) 0 9 C) 9
A 3/2 0 0 0 0 0 4
5
B af. 1 ° ° ° 0 1.0
A 6/5 a 42 2 8.4 0.4 0.6
2.5
B 416 ° 7 4 12.8 0.7 0.7
A 2/7 4 518 16 74.0 2.3 29
1.2
B 5/1 9 4b 16 40.4 1.5 27
A 4/7 6 430 16 61.4 2.3 3.2
0.6
B 6/8 1 360 nu 45.0 14 LS
A 6/6 s 192 10 32.0 17
)
(Control) 8/5 3 102 6 20.0 1.2 1.8

41


ANALYTICAL METHODS

Routine procedures (American Public Health Association, 1971) were
followed in characterizing this sewage effluent. Determinations of
dissolved oxygen (D.0.), pH, biological oxygen demand (B.0.D.), total
Kjeldahl nitrogen, total phosphates, and total, settleable, suspended,
and volatile solids were made by treatment-plant personnel at the
Silver Bay sewage treatment plant. Routine determinations of storage
temperature, pH, D.0., total hardness, total alkalinity, total acidity,
and chemical oxygen demand (C.0.D.) were generally made on each batch
of sewage. Membrane filter techniques (American Public Health Association,
1971) were used throughout this study for determination of total and
fecal coliform counts in the diluter cells and storage tanks and for
determining the degree of disinfection achieved. These bacterial
determinations were generally made two to three times weekly during
the acute and chronic tests.

The variable chemical makeup of the effluent required daily adjustments
in the chlorinated metering apparatus and air flow to the ozonators.
Total chlorine residuals were measured both colorimetrically and
amperometrically (American Public Health Association, 1971).
Colorimetric determinations were made with a Hach water test kit. The
sensitivity for determining the amperometric endpoint was significantly
increased by using a polarograph and rotating platinum electrode. The
electrode system consisted of a synchronous motor electrode rotator

and a 2,.5-cm-diameter platinum disc serving as the cathode. The anode
was composed of a 0.5-cm-diameter platinum circle placed in close
proximity to the cathode. The phenylarsine oxide titrant (5 x 107% N)
was added with a microburet to a 50-ml sample buffered to pH 4 with
sodium acetate-acetic acid and made 5 x 1073 M with potassium iodide.
The sample was mixed with a magnetic stirrer rotated slowly to minimize
turbulence at the electrode surfaces. A polarograph sensitivity of 0.1

13


Te

Table 9. HATCHABILITY OF FATHEAD MINNOW EGGS IN DECHLORINATED AND OZONATED TEST WATERS
(acute exposure system)

Techlorinated

Ozonated
Nominal
percentage Number Mean Number Mean
sewage of groups percentage of groups percentage
concentration incubated hatch Range incubated hatch Range
A 3 37.3 8-60 3 67.3 58-86
100
B 3 62.0 44-76 3 79.3 70-80
A 1 54.0 -54- 3 84.0 74-100
75
B 2 54.0 34-74 3 86.0 66-98
A 3 64.6 46-88 3 61.3 0-98
0
(Control) B 3 60.0 36-82 3 70.6 64-84



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

1. REPORT NO. 2. 3. RECIPIENT'S ACCESSION NO.
EPA-600/3-75-012
4. TITLE AND SUBTITLE

5. REPORT DATE

November 1975 (Issui
COMPARATIVE TOXICITY OF SEWAGE-EFFLUENT is: PERFORMING $75 Cssutng Date)
DISINFECTION TO FRESHWATER AQUATIC LIFE
7. AUTHOR(S:

) -

ohn W. Arthur, Robert W. Andrew, Vincent R. Mattson, [> PERFORMING ORGANIZATION REPORT NO.
Donald T. Olson, Gary E. Glass, Barbara J. Halligan,
and Charles T. Walbridge

9. PERFORMING ORGANIZATION NAME AND ADDRESS 10. PROGRAM ELEMENT NO,
Environmental Research Laboratory 1BA021; ROAP/Task 06A0J/005
Office of Research and Development |i1. CONTHACT/GRANT NO. SSS
U.S. Environmental Protection Agency
Duluth, Minnesota 55804 None (in-house)

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

Final
14. SPONSORING AGENCY CODE

Same as above

15, SUPPLEMENTARY NOTES

16. ABSTRACT Flow-through laboratory bioassays were conducted with a domestic secondary
sewage effluent that had been disinfected by chlorination, by chlorination followed
by dechlorination, and by ozonation. Effluent without disinfection served as a con-
trol. Disinfection with chlorine and ozone generally maintained the effluent at
total coliform levels of less than 1,000 per 100 ml. Lake Superior water served as
the diluent source for the experiments. Short-term (7-day) exposures were conducted
with 13 species (seven fish and six invertebrates), and long-term (generation) tests
were performed with three species (one fish and two invertebrates). In both series
of tests the chlorinated effluent was lethal at appreciably lower concentrations

than any of the other three effluent treatments. Fish were more sensitive than the
invertebrates to the chlorinated effluent in 7-day tests. The respective 7-day TL50
values of total residual chlorine to fish and invertebrates ranged from 0.08.to 0.26
and 0.21 to >0.81 mg/1. Residual ozone rapidly decreased in the treated effluent

and was not measurable in the test tanks. When special short-term test procedures
and shorter retention times for the ozonated effluent were used, measured residual
ozone was about as lethal to fathead minnows as residual chlorine. The highest mean
total residual chlorine concentrations having no long-term adverse effect on fathead
minnows, amphipods, and Daphnia were 14, 12, and 2-4 ug/1., respectively. No daphnids

survived at approximately 10 ug/1. mean total residual chlorine, a concentration that
corresponds to a chlorinated sewage concentration of about 2.5%.

n7. KEY WORDS AND DOCUMENT ANALYSIS
Pp DESCRIPTORS Ib. 1DENTIFIERS/OPEN ENDED TERMS |c. COSATI Field/Group
Disinfection Fresh water fishes Chlorine toxicity 6A
Chlorination Dechlorination Ozone toxicity 6C
Bioassay Aquatic life 7B
Aquatic animals Sewage effluent 7c
Ozone Amphipoda
Minnows
Daphnia .
Wa. DISTRIBUTION STATEMENT 79, SECURITY CLASS (This Report) 21. NO. OF PAGES
|___UNCLASSIFIED ___ n
20. SECURITY CLASS (This page) 22. PRICE
RELEASE UNLIMITED UNCLASSIFIED
EPA Form 2220-1 (9-73) 62

‘fy U.S. GOVERNMENT PRINTING OFFICE: 1975-657-695/5339 Region No. 5-1!


ABSTRACT

Flow-through laboratory bioassays were conducted with a domestic
secondary sewage effluent that had been disinfected by chlorination,

by chlorination followed by dechlorination, and by ozonation. Effluent
without disinfection served as a control, Disinfection with chlorine and
ozone generally maintained the effluent at total coliform levels of less
than 1,000 per 100 ml. Lake Superior water served as the diluent source
for the experiments. Short-term (7-day) exposures were conducted with
13 species (seven fish and six invertebrates), and long-term (generation)
tests were performed with three species (one fish and two

invertebrates). In both series of tests the chlorinated effluent was
lethal at appreciably lower concentrations than any of the other three
effluent treatments. Fish were more sensitive than the invertebrates to
the chlorinated effluent in 7-day tests. The respective 7-day TL50
values of total residual chlorine to fish and invertebrates ranged from
0.08 to 0.26 and 0.21 to >0.81 mg/l. Residual ozone rapidly decreased in
the treated effluent and was not measurable in the test tanks. When
special short-term test procedures and shorter retention times for the
ozonated effluent were used, measured residual ozone was about as lethal
to fathead minnows as residual chlorine. The highest mean total residual
chlorine concentrations having no long-term adverse effect on fathead
minnows, amphipods, and Daphnia were 14, 12, and 2-4 ug/1., respectively.
No daphnids survived at approximately 10 yg/1. mean total residual
chlorine, a concentration that corresponds to a chlorinated sewage
concentration of about 2.5%. Residual sulfite at the concentrations
used to neutralize the chlorine residual dissipated readily and had no
measurable adverse effect on the species tested.

Adi


with death. Animals were not fed during the experiments, except the
amphipods where leaves provided cover and food. Percentage mortality
of each test species was determined after 12-, 2h~, and additional
2h-hr intervals thereafter through 7 days. Tests with operculate
snails were extended for 14 days to obtain a 50% mortality level.
Standard graphical procedures were followed for determining the
concentrations resulting in 50% mortality levels (American Public
Health Association, 1971). Round stainless steel cages were used
for the stonefly and caddisfly tests. The cages were submerged

in the test tanks where the water entered and were covered with
stainless wire covers.

Routine chemical determinations were performed on the test-chamber waters.
Dissolved oxygen was generally determined every other day, and pH
analyses were made once a week on all test-chamber waters. The dissolved
oxygen measurements were made with a YSI oxygen meter. Water temperatures
were measured daily on two test chambers in each effluent treatment.

The flame photometric sodium method was used for monitoring the

nominal sewage concentrations delivered during these tests.

All residual chlorine and sulfite determinations were made on grab
samples collected at mid-tank depth with a volumetric pipette. Total
residual chlorine measurements were conducted daily during the 5-day
work week by alternating between the duplicate test chambers. Residual
ozone was not measurable in the highest concentration test-tank waters.
Residual ozone and sulfite measurements were taken in the test waters
only where additional tests were needed to establish lethal levels.

Three additional tests were conducted with the same general procedures
as above:
1) The sewage effluent was chlorinated to a residual level
of 16 mg/l. followed by neutralization with

24


For disinfecting the effluent by ozonation separate ozonators were
constructed for the acute and chronic tests. Ozone was produced by
passing dried air between concentric reactor tubes with an applied
voltage difference of 8,000 V. The voltage was obtained from a Jefferson
luminous tube outdoor transformer operated at 65 V (60 cps). The ozone
dose was adjusted by regulating the air-flow rate (98 + 18 cc/min) to
achieve a disinfection of usually less than 1,000 total coliforms per

100 ml. Details of the ozone generator are given in Figure 3. A
concentration of approximately 32 mg 03/1. in the dried air was introduced
into the bottom of a contact colum (Figure 4). Sewage effluent entered
near the top of the contact colum filled with glass marbles. The effluent
retention time in the acute and chronic ozone contact colums was 7-8

and 10-11 min, respectively. The vented air after sewage contact
contained 18 mg/1. residual ozone. To attain an effluent concentration
of 1 mg/l. residual ozone, an ozone dose rate of approximately 5.7 +1
mg/1. of sewage was needed. The disinfected effluent was then split

for delivery to the appropriate diluter.

The diluters used for the acute and chronic bioassays were patterned
after Mount and Brunge (1967)} the arrangement of diluter components was
stmilar to that given by Arthur and Eaton (1971). The percentage

sewage concentrations used for the tests are given in sections V and VI.
Vacuum manifolds from the diluters were connected in series, and the
acute and chronic diluters were separately connected to water aspirators.
Nondisinfected sewage overflowing into a plastic box (Figure 2) triggered
a microswitch, thereby opening a solenoid valve controlling the water
aspirator. This event cycled the diluters and simultaneously closed
solenoid valves delivering diluent waters. Both acute and chronic
diluter cycle rates were maintained at approximately 5 min. Flow-
splitting cells divided the test and control waters for transfer to the

duplicate acute and chronic test chambers. ‘

10


oT

Table 1. CHEMICAL CHARACTERISTICS OF THE SEWAGE EFFLUENT COLLECTED AT THE SILVER BAY, MINNESOTA,

MUNICIPAL TREATMENT PLANT

= ‘Yaar ane BeBe
zi 197
‘Jone July . ee Dees an arch | Apri ly Ok.
is z on 1 Tor eo 7 ra
Sevage flow (10" 1./day) Ja26-256y*_} 26-262) | (aa7-1s7) 136-460) | ¢a9-528) | aar-i6oy [07-13 | (203-129) | 207-253) | ca06-s36) | ca38Ra6)_| cad foo)
Iofluene
Solids (na/t.)
Sertieabie 2s 6 3.0 99 3.2 ba 2s: ae as 1 37 6a a4
(3.5515.5) | 4.0°12.09 | 4.0-11.5) | ¢6.0°18.09 | (0.62120) | (0.3-11.09 | (4.0212.0) | 65.885 9) | ¢5.6°83.09 | c2.o%45.0) | caorio.sy | ca.sci2.0) | (0213.0)
oral sar an 563 473 an 366 429 303 as ar:
(402-904) | 259-619) | (449-869) | 352-704) | (a0s-s39) | (282-460) | (366-508) | ag0°%s4) | coostzouy | cassear2y | casecerey | (320-566) | 203-305)
‘Total suspended 12 150 nue 165 108 3° 143 mn 127 36
(93-342) cat-asoy | 8-354) | 20-326) | 0-198) | (26-20) | 96-14 | Soe) | caobtdeay | cise | «23°36 cza7ey | (08-175)
‘Total suspended volatile 135 aa 180 wr 90 uy a 103 fa
c7s-i96) | (77-174) | cais-293) | coi—268) | (26-162) (80-150) | Go 7Ngey | eotzteoy | ceectezy | carve aeao | ois
1.0.0 ahd 40 49 roo | ee Lo 8 us ass uo 2 we | ws
eran | 2 (a2s-14) | casseaasy | (52-218) (112-222) | caae-242y | cuuiisey | cor-teay | (60-132) | (as-t03) | (212-106)
‘Befhvent,
Solide (as/l.)
Sertlesbie 0.6 0.2 0.2 0.2 o4
0.3 0.2 0.3 os 0.5 0.2
(0.1-1.0) | (O.1-0.4) | (0.1-0.6) | (0.1-0.9) 0.20.6) | (10.9) | (0.10.5) | (0.1-0.6) | (o.1-2.0) | @.1-1.8) | 0.11.0)
Total 26 2 290 us 216 256 m2 265 209 an ue
¢2s6-380) | (#5-300) | (240-588) | 208-279) | (236-306) | (210-202) | (213-2679 | agd Zea) | casedary | carsedasy | caostzesy | cantasr | aricesey
Forel suspended 4 % 1 20 26 7) u as a %
casey | cas-60) | caeeay | ae40y | ars | aia | air | ashe) ery | erin | aes axa | eas)
‘Total auspended volatile 38 » By 2% 20 a 2» x0 3 n
arty | artsy | ao | aoa | asm | on | are | ay | ase | arkn | ast | aoke | aan
3.0.0 (ag/1-) 40 29 23 3 32 4 32 3 | so 60 48 Bs 25
qs | asa | asap | cz0-30) | cases) | cas-6oy | c2t-90) | casmasy!eaecsey | cena | cams) | aoreay | as-27)
Dissolved oxygta (ng/1,) 5.5 6.0 $7 5.0, 3.2 3.7 55 6a 6.2 6.9 7 5.2 5.6
* 6.56.3) | 6.56.5 | 6.25.0) | aes.) | GIy | 6.25.9) | 6.255. | 6.7569) | 56.6 | 69:8. ] 0.99.5) | boar | 6.05.9)
pa (untes) 24 13 22 Ta 12 7a ra 6.9 69 73 1
27.6) | @.0-7s) | 07) | dra | aoa) | 7.07.9 | 7.027.2) (6.8-7.0) | 6.77.0) | 7.27.3) | @.257.5)
Kjeldant nterogen (ag/1.) 15.1 4.5, 11. 13.7 13.6 13.6 16.1 19.5 18.4 20.6 rey 3 14.0
14,8515040 | (42,5-25.4) | (0,612.3) | 5-17.19 | e.3-47.6) | c2.9-15.2) | 16.0°19.0) | as.eczzay | caa.2-22.8) | 7-26.39} (1.2217.4) 36.5)| c1.8-15.1)
‘Total phosphates (as TO, eg/i.) | 11.0 aa 10.2 23 13 13.2 13.8 30.5 10.3
(8.5-13.3) 20,8-17.3) 6.5512.0) 4.29.3) | 3540.29 | a2.i4i7.0)| a1.6i9.0) (8,811.6) (9.0212.3)

hange 40 parentheses.


To Gam. To FH.
diuter Giuter

*

Figure 4. Apparatus used for volumetrically dividing and ozonating
the sewage effluent in chronic tests.

12


sulfur dioxide gas. Any associated toxicity from this
procedure was determined with fathead minnows at a test
temperature of 18° + 1° C.

2) Tests were also conducted on the concentrations of sulfite
lethal to fathead minnows and amphipods. The SO gas was
added to the nonchlorinated effluent. The test water
temperature was 19° + 1° C.

3) Fathead minnow eggs were obtained from control waters in the
chronic test and incubated in the two highest dechlorinated
and ozonated test concentrations (100 and 75%) and controls.

Procedures to determine hatchability differences followed the
recommendations of the National Water Quality Laboratory (1971). The
test water temperature was 24° + 1° C,

Two tests were also conducted to determine the concentrations of residual
ozone lethal to fathead minnows under flow-through conditions. Special
procedures were used for this test with the ozonated effluent. To
maintain a residual the ozonated sewage was conveyed directly from the
contact column to glass boxes each holding 650 ml. The boxes were
arranged in a five-step sequence, in which ozonated sewage flowed from
one box to the next. A diluter was not used for this test. Five
fathead minnows were placed in each of the glass boxes and tested at

a temperature of 23° + 1° C. Test concentrations were not duplicated.

RESULTS

After 7 days the chlorinated effluent was appreciably more toxic to the
test animals than the other effluent treatments tested (Table 7). More
than 50% of the fish tested died in chlerinated effluent concentrations
of 12-37%. Less than 50% of the operculate snails died in the

highest concentration of chlorinated effluent. Mean mortality in

the two highest dechlorinated and ozonated concentrations (100% and 75%)
was less than 50% and 20%, respectively. In the highest nondisinfected

25


aN

(to te temp) (to a temp)
[ ] | |
Nondisinfected Ozonation Ozonation Nondisinfected
Waste Column Column Waste
Chlorination Chlorination
Contact Cell Contact Cell
Dechiorination Dechtorination
» Contact Cell Contact Cell
Fy ry Fy
A B A B A B A B
LU______. Acute bioassays ——__--__! LL Chronic bioassays

A-Fathead minnow tests
8B-Amphipod and Daphnid tests

Figure 1. Sewage flow from storage tanks to diluter cells.


Nightingale, H. W. 1931. Ozone treatment of waste sulphite liquor with
reference to the effects upon cutthroat trout, chinook salmon, and oysters.
Pacific Pulp Paper Ind. 5:23-28.

Perkin-Elmer Corporation. 1971. Analytical methods for atomic absorption
spectrophotometry. Norwalk, CT.

Seligson, D. and H. Seligson. 1951. A microdiffusion method for the
determination of nitrogren liberated as ammonia. J. Lab. Clin. Med.
38:324-330.

Shuval, A. I., J. Cohen, and R. Kalodney. 1973. Regrowth of coliforms
and fecal coliforms in chlorinated wastewater effluent. Water Res. 7:
537-546.

Sliter, J. T. 1974. Ozone: An alternative to chlorine? J. Water
Pollut. Control Fed. 46:4-6.

Tsai, C. 1973. Water quality and fish life below sewage outfalls.
Trans. Amer. Fish. Soc. 102:281-292.

U.S. Environmental Protection Agency. 1973. Secondary treatment
information, notice of proposed rulemaking. Federal Register
38:10642-10643.

Zillich, J. A. 1972. Toxicity of combined chlorine residuals to
freshwater fish. J. Water Pollut. Control Fed. 44:212-220.

51


10

11

12

Acute Toxicity of Chlorinated Effluent to
Seven Fish and Six Invertebrate Species

Hatchability of Fathead Minnow Eggs in
Dechlorinated and Ozonated Test Waters

Survival and Reproduction of Fathead Minnows
During 43 Weeks' Continuous Exposure to
Nondisinfected and Disinfected Effluents

Survival and Reproduction of Amphipods after
20 Weeks' Continuous Exposure to Nondisinfected
and Disinfected Effiuents

Survival and Reproduction of Daphnia magna During

2 Weeks’ Continuous Exposure to Nondisinfected
and Disinfected Effluents

vili

31

37

41

43


SECTION V

ACUTE TESTS
METHODS

Modified proportional diluters served as the water~delivery system for
each of the four effluent treatments. The disinfection apparatus and
the diluters are described in section IV. The nominal percentage
sewage concentrations used for the nondisinfected, dechlorinated, and
ozonated treatment tests were 100, 75, 50, 25, and 12%. For the
chlorinated effluent tests, nominal sewage concentrations were set at
one-half of the other three treatments. The diluent source was raw
Lake Superior water piped directly to the laboratory. Dupliicate test
chambers were used for the five effluent concentrations and the controls.
Diluter cycle rates were maintained at approximately 5 min throughout
the tests. The test tanks held 15 1. of water and had a water-retention
time of approximately 5 hr. Durotest (Optima FS) and widespectrum
Gro-lux fluorescent tubes provided the light source, and a constant

16-hr photoperiod was used.

Acute exposure tests were conducted with seven fish and six invertebrate
species. Sources and sizes of the test animals are given in Table 6.
Before testing, all animals were acclimated in laboratory holding tanks
for 10 days or more within 2° C of test temperature. Ten organisms
randomly selected were introduced to each test tank so that 20 individuals
of each species were tested at each concentration. Any damaged or
diseased individuals were rejected before testing. Complete
immobilization of the organisms was considered the endpoint and equated

22


Appendix Table 5. NUMBER OF SURVIVING FATHEAD MINNOWS
AFTER 19, 25, AND 43 WEEKS' CONTINUOUS EXPOSURE TO

NONDISINFECTED AND DISINFECTED SEWAGE EFFLUENT

Nominal Nominal
percentage a 5 percentage b
sewage 10’ 25' 43 sewage 10° 25"
concentration | weeks |weeks |weeks | concentration | weeks | weeks
Nondisinfected Dechlorinat
a 35 15 u A 24 B 9
20 20
B 29 2 10 B 23 2 2
A 26 1 5 A 24 b 4
10 10
B 27 1s 9 B 25 23 | 10
A 28 5 190 rN 20 Bb uu
5 5
B ce uu 10 B 25 1b 2
A 27 a 9 A 23 B 10
2.5 25
B 23 n 9 B 26 B 10
A 23 n 8 A 27 14 10
1.2 12
B 28 sy B B 28 3 5
A 27 u 8 A 26 n 9
° 0
(Control) B 29 ph 10 (Control) B 25 13 phe
Chlorinated” Qzonated
A 8 2 2 A 36 15 10
10 20
B 1 1 1 B 30 15 10
A 5 5 5 A 30 Rn 9
5 10
B 4 4 5 B 23 15 10
aA 20 B 10 A 23 13 14
2.5 5
B 19 15 9 B 32 B 9
A Lb ub 3 A 30 ary 13
12 2.5
B BR 10 9 B 23 uw B
A 3 13 9 A 26 u 9
0.6 12
B 20 uw un B 20 15 2
A 18 n 7 A 31 B 10
0 a
(Control) 8B 2h 15 13 G@ontrol) B 3 22 5

“gach test chamber (N = 35) thimned to 15 fathead minnows after 60 days.

Suxcess males removed during the spawning perfod.

“Respective total resfdual chlorine levels (tm ug/l.) were 110, 42, 14, 6, 3,
and noumeasurable.

58


Appendix Table 7. NUMBER OF SURVIVING AMPHIPODS AFTER 4, 10, 16, and 20 WEEKS’
CONTINUOUS EXPOSURE TO NONDISINFECTED AND DISINFECTED SEWAGE EFFLUENT

Nominal Nominal
percentage a percentage
sewage 4 10 16 20 sewage 4 108 16 20
concentration | weeks | weeks | weeks | weeks|| concentration | weeks | weeks | weeks | weeks
Nondisinfected Dechlorinated

A 25 22 12 10 A 10 u 7 7

20 20
B 20 15 5 1s B wu wu 1 0
A 22 20 wu 10 A uv 2 2 R

10 10
B 29 29 15 5 B 19 21 2 n
A 20 2 1B 12 A 28 a7 | w n

5 5
B 23 19 a 15 B 23 20 un 4
A 19 wv ey 12 A 27 | 29 wb 15

2.5 2.5
B 30 28 10 10 B 16 wu as. | a7
A 15 19 13 ey A 27 26 3 12

1.2 1.2
23 18 10 u B 24 23 cry ub
A 24 2 u 2 A 22 20 13 14

C) °
(Control) B 27 32 n 11 || Gontrol) B 28 25 13 B

Chlorinated” Ozonated

A 2 9 ° o b 18 we 13 m

10 i 20
Bol 24 i0 x 9 B 16 18 14 13
A 29 20 6 5 A 30 28 10 8

5 10
B 25 19 6 5 B 2 1g 9 u
A. 21 23 i u A 23 uv 2B u

2.5 5
B 27 25 10 10 B 16 16 10 12
A 22 19 10 9 A 31 21 uw 2B

12 2.5
25 25 4 16 B 25 26 12 2
A 28 20 12 u A 21 20 15 ub

0.6 1.2
B 9 13 15 Ty B Ww Bb |p 10
A 24 22 10 12 A 16 22 13 12

° C)
(Controt) B 25 21 15 13 || @ontrol) 8 22 21 14 13

gach test chamber (N= 35) thinned to 15 amphipods after 60 days.

respective total residual chlorine levels (in ug/l.) were 123, 54, 19, 12, 8, and nonmeasurable.

60


T9

Appendix Table 8.

NONDISINFECTED AND DISINFECTED SEWAGE EFFLUENT

SURVIVAL AND GROWTH OF SECOND GENERATION AMPHIPODS AFTER 30 DAYS' CONTINUOUS EXPOSURE TO

Nondisinfected Dechlorinated Ozonated Chlorinated®
Nominal ‘Average ‘Average Average Nominal ‘Average
percentage final head final head final head percentage final head
sewage Percentage length Percentage length Percentage ‘length sewage Percentage Length
concentration survival (on) survival (am) survival (oa) concentration survival (wn)
7
A 90 0.6 88 0.7 65 0.8 A as? 0.7
20 10 b
B 100 0.6 66 0.6 85 0.8 B a2 0.6
A 88 0.6 89 0.7 9s 0.8 A 96° 0.8
10 5 b
B 98 0.6 a 0.6 98 0.8 8 95 0.7
A 58 0.5 97 0.7 86 0.8 A 50 0.7
5 2.5
B 100 0.6 98 0.7 75 0.5 B 85 0.7
A 53 0.5 91 0.7 86 0.7 A 90 0.6
2.5 1.2
B 96 0.6 90 0.7 75 0.7 B 92 0.7
A 93 0.7 90 0.7 67 0.6 A 86 0.6
1.2 0.6
B n 0.6 91 0.7 93 0.7 B 92 0.6
A 70 0.6 89 0.6 37 0.6 A 95 0.6
o o
(Control) B _ _ 100 0.5 88 0.6 (Control) B 19 0.6

"wean total residual chlorine levels for the six concentrations (in ug/l.) were 135, 65, 17, 4, 2, and nonmeasurable, respectively.

best animals taken from control tanks.

“Test not conducted.


measurements and ozone column measurements were generally made weekly.
Substantial amounts of residual ozone were lost in passage from the
ozone contact column to the diluter cells (Table 3). Approximately 50%
less total residual chlorine was measurable in the chlorinated diluter
cells by the orthotolidine method than by the amperometric method.

Excess total residual chlorine was not measurable in the dechlorinated
effluent during any of the acute tests despite variations in the residual
sulfite levels. On three occasions residual chlorine was measurable in

the dechlorinated effluent for the chronic tests (see section VI).

The mean concentrations of total coliforms in the effluent before and

after disinfection are given in Tables 4 and 5. After disinfection

the mean coliform concentrations were less than 10,000 per 100 mi in

13 of 15 acute tests. About 10 times more coliforms were present in

the dechlorinated than in the chlorinated effluent. Coliform concentrations
were generally higher in the ozonated effluent than in the chlorinated
effluent during the acute tests. During the fathead minnow and amphipod
chronic tests all mean total coliform concentrations after disinfection
were less than 10,000, and after the eighth week concentrations of coliforms
were generally less than 1,000 per 100 ml. A loss in measurable coliform
bacteria occurred during the flow of effluent from the storage tank

to the nondisinfected diluter cells (Tables 4 and 5); no single

component of the system was largely responsible. In the Daphnia

chronic tests the mean total coliform concentrations after disinfection
were <1,000 per 100 ml.

Disinfection of fecal coliforms in the acute and chronic test systems

was greater than 99.9%. Mean fecal coliform counts were generally
less than 10 per 100 ml in the treated effluents.

19


CONTENTS

Abstract

List of Figures
List of Tables

Acknowledgments

Sections

I
II
III
Iv

VII
VIII

Conclusions
Recommendations
Introduction

General Bioassay and Analytical Procedures

Disinfection
Analytical Methods
Results
Acute Tests
Methods
Results
Chronic Tests
Methods
Results
Fathead Minnow Chronic Test
Amphipod Chronic Test
Daphnia Chronic Test
Discussion
References
Appendices

DAP WR

15
22
22
25
32
32
35
36
39
40
44

49
52


This report was submitted in fulfillment of Project Numbers 18050 WAP
and 16AAE by the Environmental Research Laboratory-Duluth (formerly

the National Water Quality Laboratory). Experimental work was completed
in November 1972.

iv


A series of glass chambers (after Brungs and Mount, 1970) was designed
to separate the nondisinfected effluent from that being chlorinated and
dechlorinated. Details of this apparatus employed for the chronic tests
are shown in Figure 2. For chlorination, distilled water from a Mariotte
bottle entered near the bottom of a glass tube. The bottom of the glass
tube was equipped with a fritted glass disc for dispersing the entering
chlorine gas. This saturated solution of chlorine was added to the
nondisinfected effluent by a chemical-metering apparatus (Mount and
Brungs, 1967). The concentration of chlorine in this solution was
maintained at 3.5 + 0.6 mg/ml. The chlorine contact chamber had a
retention time of approximately 30 min. At the end of this chamber the
chlorinated effluent was again separated for dechlorination and for
delivery to the chlorinated effluent diluter. Sulfur dioxide gas was
used for dechlorination with a chemical-metering apparatus similar to
that used for chlorination. At the end of the dechlorination contact
cell (approximately 40-min retention time) the effluent flow was split
for delivery to the appropriate diluter.

The basic difference in the equipment used for acute tests was the
absence of modified diluters at the end of the dechlorinated contact
cell. A wooden enclosure surrounded each acute and chronic disinfection
apparatus for venting the surrounding air to the outside of the
laboratory. The chlorine dose was adjusted so that a total residual of
approximately 1 mg/1. (orthotolidine colorimetric determination) remained
after a 30-min retention time, which would result in a total coliform count
of less than 1,000 cells per 100 ml. To maintain this residual, 3.8 +
0.4 mg Cl/1. of sewage was used. The sulfur dioxide (dechlorination)
dose rate was adjusted to neutralize the chlorine residual and provide an
excess sulfite residual of approximately 4 ppm in case of an overdose

of chlorine.


TABLES

Chemical Characteristics of the Sewage Effluent
Collected at the Silver Bay, Minnesota, Municipal
Treatment Plant

Chemical Characteristics of the Sewage Effluent
in the Laboratory Storage Tanks

Total Residual Chlorine and Ozone Concentrations
Measured from Diluter Toxicant-Bearing Cells and
Ozone from Column During the Acute and Chronic
Tests

Concentrations of Total Coliforms in the Storage
Tanks and Diluter Cells During the Acute Tests

Concentrations of Total Coliforms in the Storage
Tanks and Diluter Cells During the Chronic Tests

Sources and Sizes of Animals Used for the Acute
Tests

Mortality of Seven Fish and Six Invertebrate
Species in Acute Tests of Various Concentrations
of Four Effluent Treatments after 7 Days’

Exposure

vil

17

18

20

21

23

26


ua/mm (10-25 wa full scale) provided sufficient discrimination of the
endpoint with additions of titrant as small as 0.01 ml. Ozone
concentrations were determined volumetrically by using the same
procedures. For both the residual chlorine and ozone measurements,
Samples were titrated immediately after the iodide and buffers were

added. Complete descriptions of the analytical procedures are given by
Andrew and Glass (1974).

Sulfur dioxide concentrations were measured as sulfite by using a

solution of potassium iodate, 5 x 10" N, as the titrant and the
amperometric method already described to detect the endpoint. The

sample to be titrated was made approximately 0.2 M H2S0, and 5 x 107? M KI.
As titrant is added the sulfite is oxidized to sulfate, and an excess
converts iodide to iodine for amperometric detection. Since iodide in

the presence of oxygen is oxidized gradually at low pH, the titrations
were performed within 3 min after the samples were taken.

Ammonia was determined colorimetrically with Nessler reagent and a
microdiffusion concentration technique (Seligson and Seligson, 1951).
In this procedure a 2-ml sample was placed into a 50-ml sample bottle,
2 ml of saturated K,CO3 solution was carefully pipetted into the bottle
to form a layer under the sample, and the bottle was then quickly
stoppered with a receiving rod whose ground glass tip had been coated by
dipping into 0.5 M H)S0,. The ammonia diffused out of the sample and
onto the acid-coated rod tip as the sample bottle was rotated
horizontally at 50 rpm for 30 min. The receiving rod was transferred
into a colorimeter tube containing 10 ml. of Nessler reagent, mixed,
and after 10 min the optical absorbance at 420 nm was determined.
Standards over the range of 5-30 ug ammonia (as Ng) were used.

14


EPA-600/3-75-012 ; -
November 1975 Ecological Research Series



Table 10. SURVIVAL AND REPRODUCTION OF FATHEAD MINNOWS DURING 43 WEEKS'

CONTINUOUS EXPOSURE TO NONDISINFECTED AND DISINFECTED EFFLUENTS

Nominal Final Number of Egg Hatchability
percentage number excess males| Total eggs | Eggs Spawnings Number Mean
sewage surviving Temoved and per per of percentage
concentration | (male/female) (Nov-Jan) spawnings | female | female spawnings hatch
Nondisinfected

A al 3 6817 (50) 974 7 22 66

20
B 3/5 3 7377 (60) | 1475 12 2 53
A aft 5 244 (2) 246 2 1 80

10
B 4/5 7 aeos (64) | 1762 13 14 64
A 416 5 7922 (64) | 1304 n wv 82

5
B 416 1 10586 (90) | 1764 15 15 78
A 45 4 11546 (88) | 2309 18 vv 59

2.5
B 415 2 9407 (62) | 1881 2 11 m1
A als 2 5426 (62) | 1357 16 16 nR

12
3/10 ° 16524 (132) | 1652 13 22 61
A 41h 4 5950 (64) | 1488 16 wu 84

°
(Control) B 5/5 1 9148 (74) | 1830 15 12 69

Chlorinated”

A a 0 o @ a 0 _ _

10
B o/1 0 a @) Q 9 _ —_—
A 3/2 0 7435 (55) | 3717 28 ry 5

5
B 3/2 0 4833 (29) | 2416 ww 9 20
A 416 2 7203 (72) | 1200 2 26 86

2.5
B 45 4 7193 (73) | 1439 45 22 87
A 4/9 2 7661 (74) a51 8 18 83

1.2
B 3/6 ° 9626 (97) | 1604 16 19 75
A 4/5 4 7550 (69) | 1510 1b 18 a1

0.6
B 3/8 3 3726 (36) 466 4 13 as
A 413 3 8250 (68) | 2750 23 15 83

C)
(Control) 8 419 1 5038 _ (35) 360 4 1. mL

37


1.

2.

3.

4.

5.

6.

SECTION I

CONCLUSIONS

Chlorinated effluent was lethal to the test animals at appreciably
lower concentrations than nondisinfected, dechlorinated, and
ozonated effluents in both short- and long-term tests.

Both ozone and chlorine generally maintained effluent disinfection
levels at <1,000 total coliforms per 100 ml.

Residual ozone rapidly disappeared from the effluent and was not
measurable in the test-tank waters.

During the short-term tests fish mortality was more rapid than
invertebrate mortality in the chlorinated effluent.

The highest mean total residual chlorine concentrations having no
long-term adverse effect on fathead minnows, amphipods, and Daphnia
were 14, 12, and 2-4 ug/l., respectively. This corresponds to a
concentration of 1.2 to 2.5% chlorinated effluent.

The lowest mean total residual chlorine concentrations having a
measurable long-term effect were 42 yg/1. for fathead minnows
(survival of initial animals), 19 yg/1. for amphipods (reproduction),
and approximately 10 yg/1. for Daphnia (survival of initial animals).


ACKNOWLEDGMENTS

We would like to thank the staff of the Silver Bay, Minnesota, Sewage
Treatment plant for their generous support in providing equipment and
giving time for the effluent collections. Appreciation is expressed to
many members of the Environmental Research Laboratory-Duluth (formerly
the National Water Quality Laboratory) staff who provided assistance
during the course of this project. Mr. F. A. Puglisi devised and
constructed the special chemical metering apparatus and determined the
dose concentrations. Mr. G. R. Endicott designed and constructed many

of the supportive components incorporated into the disinfection and
bioassay process. Bacterial analyses were performed by Ms. S. Bagley,

Mr. D. Herman, and Mr. R. M. Brosdal; Ms. V. Snarski and Mr. L. F. Mueller
performed the routine analyses and contributed bioassay assistance. Mr.
A. R. Carlson assisted with the acute tests. The effluent was transported
to the laboratory and maintained in the storage tanks by Mr. D. Beedy and
Mr. R. E. Lewis. Dr. T. W. Thorslund performed the statistical analyses
reported in section VI. In addition, we thank Ms. S. L. Forseth for
typing this manuscript.

ix


Dunnett, C. W. 1955. A multiple comparison procedure for comparing
several treatments with a control. J. Amer. Stat. Assoc. 50:1096-1121.

Dunstan, W. M. and D. W. Menzel. 1971. Continuous cultures of natural
populations of phytoplankton in dilute, treated sewage effluent. Limnol.
Oceanogr. 16:623-632.

Esvelt, L. A., W. J. Kaufman, and R. E. Selleck. 1973. Toxicity
assessment of treated municipal wastewaters. J. Water Pollut. Control
Fed. 45:1558-1572.

Hubbs, C. L. 1930. The high toxicity of nascent oxygen. Physiol. Zool.
3:441-460.

Institute of Maritime Fisheries. 1972. Use of ozone in sea water for
cleansing shellfish. Effluent and Water Treat. J. 12:260-262.

Kelly, C. B., W. Arcisz, and M. W. Presnell. 1960. Bacterial
accumulation by the oyster Crassostrea virginica, on the gulf coast. U.S.
Dep. Health, Education, and Welfare, Public Health Service, Robert A. Taft
Sanitary Engineering Center Tech. Rep. F60-4. 26 p.

MacLean, S. A., A. C. Longwell, and W. J. Blogoslawski. 1973. Effects of
ozone-treated seawater on the spawned, fertilized, meiotic, and cleaving
eggs of the commercial American oyster. Mutation Res. 21:283-285.

Michigan Department of Natural Resources, 1971. Chlorinated municipal
waste toxicities to rainbow trout and fathead minnows. U. S. Environmental
Protection Agency, Washington, D.C, Water Pollut. Control Res. Ser.,
EPA~18050GZZ10/71, October 1971. 49 p.

Mount, D. I. and W. A. Brungs. 1967. A simplified dosing apparatus for
fish toxicology studies. Water Res. 1:21-29.

National Water Quality Laboratory. 1971. Recommended bioassay procedure
for fathead minnow Pimephales promelas Rafinesque chronic tests. Inc. I.
Weber (editor), Biological field and laboratory methods for measuring the
quality of surface waters and effluents. U.S. Environmental Protection
. Agency, Cincinnati. Env. Monitor. Ser., EPA-670/4-73-001, July, 1973.

Nebel, C., R. D. Gottschling, R. L. Hutchinson, R. L. McBride, D. M.
Taylor, J. L. Pavoni, M. E. Tittlebaum, H. E. Spencer, and M. Fleischman.
1973. Ozone disinfection of industrial - municipal secondary effluents.
J. Water Pollut. Control Fed. 12:2493-2507.

50


Adult amphipods, Gammarus pseudolimnaeus Bousfield, were collected from
the Eau Claire River near Gordon, Wisconsin. Fully gravid females were
isolated in the laboratory and 35 newborn young, 1-6 days old, were
randomly placed on June 25, 1971, into each of 48 progeny tanks holding
7.4 1. of water. Test temperature was maintained at 17.5° + 1° C.

Mean retention time for the progeny chambers was approximately 4 hr.
After 60 days the amphipods were transferred to corresponding adult
tanks with a water capacity of 14.7 1. and impartially thinned to 15
individuals per tank. The photoperiod was the same as that used for the
fathead minnow chronic test. The amphipods matured to adults in early
September and started producing young that continued until test
termination on November 10-11, 1971. Measurements of head capsule length
after 30 days' exposure in the progeny chambers were used for determining

growth of the second generation young.

The diluters and test chambers housing the fathead minnows and amphipods
were separately encompassed by black plastic enclosures to minimize
external physical disturbances. Laboratory air temperatures generally
ranged from 17° to 24° C. The amphipod enclosures were air conditioned to
reduce air supersaturation in the test waters. However, during the 14th
week of the test air bubbles were observed clinging to the external
surfaces of the amphipods, making them more buoyant. Aeration of the test
water during this time eliminated the air-bubble problen.

Daphnia magna for the chronic tests were obtained from laboratory cultures.
Test temperature was maintained at 22° + 1° C. The adult amphipod chambers
and the same mean water-retention times were used for this test. Ten
1-day-old (16-24-hr) daphnids were placed into each chamber for tests

of 2 weeks' duration during July and October 1972. To maintain the
daphnids in the control tanks, food was continuously added with a
peristaltic (Durum Co.) pump to the diluent water head box. Food source
used for the July test was a 20:1 (31.5 g) mixture of Glencoe No. 2 trout

33


a4

Table 5. CONCENTRATIONS OF TOTAL COLIFORMS IN THE STORAGE TANKS AND DILUTER CELLS DURING THE CHRONIC TESTS
(mean 4~week log counts per 100 m1)

“test organism Storage tank Nondisinfected Dechlorinated Chlorinated Ozonated

and time (weeks) |Hean log) N | Range | Mean log |N | Range [Mean log Range | Mean log|N | Range | Mean log) N | Range

Fathead mlnnows
0-4 5.46 | 4|5.28-5.79 | 5.51 | 4|5.11-5.76 | 3.95 | 4] 3,48-4.45 { 2.20 | 4] 1.72-2.58 | 3.68 | 4| 2.87-4.91
5-8 5.82 | 915.43-6.49] 5.46 | 9 |4.85-5.86 | 3.58 | 9] 2.15-4.85 | 2.02 | 9] 1.08-2.38 | 2.79 | 9| 2.15-3.32
9-12 5.57 | a1] 5.15-6.27 | 5.43 Jan ]4.05-6.45 | 2.76 | 11] 0-426 | 1.51 | 11] 0-2.66 2.55 | 11| 1.48-3.48
13-16 5.62 | 9{5.15-7.00 | 5.36 | 9 }4.e5-5.56 | 1.89 | 9|3.04-1.48 | 1.60 | 9{2.17-0.90 | 2.05 | 9|1.48-3.46
17-20 5.63 | 6|4.95-6.30 | 5.16 | 6 |4.67-5.91 | 1.70 | 6]1.00-2.62 | 1.87 | 6|1.20-2.62 | 2.12 | 6] 1.00-3.76
21-24 5.29 | 10] 4.85-5.88 | 4.99 10 |4.48-5.84 | 1.91 | 10/1.00-3.81 | 1.25 | 10]0.60-2.26 | 1.75 |10] 0-3.15
25-28 5.53 | 22] 5.21-5.93 | 5.32 |a2 ]4.70-5.79 | 1.75 }12) 0-4.32 | 1.51 | 12] 0.60-2.40 } 1.30 }12] 0-2.08
29-32 5.51 | 9[4.70-6.05 | 5.25 | 9 [4.48-5.99 | 2.42 | 9] 0-2.36 | 1.72 | 9]0.60-2.57 | 1.57 | 9| 1.00-2.53
33-36 5.56 | 10| 4.60-6.26 | 5.07 10 ]4.00-5.74 | 1.67 | 10] 0-2.59 | 0.93 |10} O-1.51 | 1.56 | 10] 1.00-2.26
37-40 5.66 | 9] 4.30-6.77 | 5.36 | 9 |4.00-6.53 | 1.66 | 9} 0-3.73 | 1.45 | 9] 0-409 | 2.23 | 9] 0-4.21
41-43 5.64 | 7|4.60-6.64 | 5.59 | 7 |4.60-6.56 | 1.76 | 7| 0-3.04 | 1.45 | 7] 0-3.08 | 2.56 | 7| 2.08-3.15
O-4 5.72 | 9|5.18-6.08 | 5.33 | 9 |4.78-5.72 | 4.00 | 9{2.63-4.84 | 2.24 | 9]1.60-2.91 | 2.83 | 9 | 2.28-3.52
5-8 5.70 | 9{5,32-6.41 | 5.39 | 9 [4.90-6.04 | 3.28 | 9/2.85-3.90 | 1.29 | 9] 0-2,00 | 2.56 | 9] 2.04-3.65
9-12 5,35 | 8/4.85-5.73 | 4.97 | 8 ]4.30-5.52 | 1.34 | 8] 0-2.00 | 0.26 | 8] 0-0.90 | 1.58 | 8] 1.30-3.54
13-16 5.65 | 8|5.20-6.12 | 5.44 | 8 j5.08-5.73 | 1.92 | 8] 1.48-2.72 | 1.51 | 8}0.60-2.23 } 1.96 | 8] 1.30-3.54
17-20 5.33 | 8] 4.78-5.92 | 5.01 | 8 |4.30-5.61 | 1.79 | 8]1,00-2.20 | 1.13 | 8]0.60-1.64 | 1.43 | 8]  0-4.32



Table 10 (continued). SURVIVAL AND REPRODUCTION OF FATHEAD MINNOWS DURING

43 WEEKS' CONTINUOUS EXPOSURE TO NONDISINFECTED AND
DISINFECTED EFFLUENTS

Nominal Final Number of Egg Hatchability
Percentage number excess males| Total egge | Eggs Spawnings | Number Mean
Sewage surviving removed and per per of percentage
concentration | (male/female) | (Nov-Jan) spawnings | female | female bpawnings hatch
Dechlorinated
1 A 4s 4 17579 (114) | 3516 23 a 48
0
B 3/9 0 12480 (109) | 1387 2 25 43
A 4/10 0 9238 (70) 924 7 12 3
10
B 416 3 10099 (77) | 1683 13 25 2
A 4/7 2 17110 (103) 2644 15 25 66
5
B 4/8 3 7272 (75) 909 9 20 82
A 416 1 10291 (99) | 1715 16 28 B
2.5
B 416 1 7604 (62) | 1267 10 12 79
A 416 4 701s (63) | 1170 10 18 84
1.2
B aft 4 1297 (12) | 1297 2 3 60
a 4/5 o aus (78) | 1684 16 18 69
0
(Control) B 5/6 2 3654 (26) 609 4 2 87
Ozonated
A 416 5 7033 (77) | 1172 13 14 n
20
B 416 5 8595 (81) | 1432 4 1s 87
A 5/4 3 8039 (71) | 2010 18 7 £3
10
B 46 4 8776 (67) | 1463 n 13 60
A 5/9 L 8175 (78) 908 9 15 3
5
B 45 3 10001 (101) | 2000 20 wv 73
a 6/7 ° 8822 (91) | 1260 13 18 75
2.5
B 5/8 1 5472 (56) 684 7 uv 5h
A 4/5 4 6342 (64) | 1268 1B 1b 53
L2
B 4/8 4 9802 (89) | 1225 nu as n
A 5/5 3 7061 (86) | 1412 uw 15 6
°
(Control) _B 4p. 4 4705_ (25) __|_ 4705 25 10 82

@Initial number of fathead minnows per tank was 35, thinned to 15 individuals after 60 days.

Pyean total residual chlorine levels for the six test concentrations (in ug/l.) were 110, 42, 14, 6, 3, and
nonmeasurable, respectively.

38


0z

Table 4,

CONCENTRATIONS OF TOTAL COLIFORMS IN THE STORAGE TANKS AND DILUTER CELLS DURING THE ACUTE TESTS
(mean log counts per 100 m1)

Storage tank Nondisinfected Dechlorinated Chlorinated zonated
Test organien ‘Mean log [_W ‘Range. Wean log Range Mean log [_N ‘Range Mean Jog { W Range Mean logi 6 Range
vu .
Brook trout 5.76 | 6 | 5.45-6.07] 5.49 | 6 | S.11-5.68] 3.96 | 6 | 3.60-4.42} 3.22 | 6 | 0.60-3.63] 2.41 | 6 | 2,08-2.78
Coho salmon 5.91 | 6 | 5,436.49] 5.71 | 6 | 5,536.01] 3.97 | 6 | 3.36-5,31] 3.07 | 6 | 2.41-3.79] 3.24 | 6 | 2.51-4.20
Fathead minnow 6.02 | 5 | $.30-6.40] 5.59 | 6 | s.oe6.8| 4.02 | 5 | 3.60-6.43] 6.26 | 5 | 402-456] 3.98 | 6 | 3.49-6.76
Fathead minnow 5.86 | 6 | 5.62-6.30] 5.83 | 6 | 5.30-6.05 4 _ . 2.01 | 6 | 1.60-2.27 _ _ -
White sucker 5.40 |] 6 | 5.15-5.76] 5.26 | 6 | 495-541] 2.99 | 6 | 2.62-3.59] 1.95 | 6 | 1.60-2.29] 1.54 | 6 | 1.00-2.10
Walleye 5.72 | 6 | 5.40-6.27] 5.63 | 6 | 5.30-6.09] 3.33 | 6 | z.91-4.19| 2.15 | 6 | 1.30-3.22] 2.69 | 6 | 2.18-3.66
Yellow perch 5.82 | 6 | 5.34-7,00/ 5.59 | 6 | 4.85-7,05] 2.87 | 6 | 2.66-3.11/ 1.66 | 6 | 1.45-1.88/ 2.36 | 6 | 1.3074.09
Largemouth bas: 5.44 | 6 | 4,855.94] 5.50 | 5 | 5.00-5.83/ 2.68 | 5 | 2.26-3.04] 1.40 | 6 | 1,081.83) 2.03 | 6 | 1.48-3.63
Invertebrates
‘Amphipod 6.51 | 6 | 5.63-7.20] 6.18 | 6 | 5.85-6.41| 4.26 | 6 | 3.28-5.52] 3.34 | 6 | 3.18-3.54] 3.82 | 6 | 3.34-4.24
Amphipod 5.63 | 6 | 4.95-6.13] 5.44 | 6 | 4.90-6.05 _ _ _ 1.99 | 6 | 1,56-2.46 _ . _
Stonefly 5.44 | 5 | 4.95-5.88] 5.31 | 5 } 4.85-5.09] 2.77 | 5 | 2.40-3,85] 1.58 | 5 | 1.20-1.94] 1.99 | 5 | 1.00-2.36
Caddisfly 5.46 | 5 | 4.85-5.91]} 5.57 | 5 | 5.15-5.86 _ _ _ 2.29 | 5 | 0,90-4.29 - - _
Crayfish 5.49 | 6 | 5.25-5.76} 5.21 | 6 | 4,785.46) 9 _ _ _ 1.43 | 6 | 0,60-2.00 _ _ _
Operculate snai: 5.80 9 4.95-6.36 5.73 9 4.90-6.94 2.83 9 2.08-3.67 1.99 9 1,60-2.27 2.28 9 1.00-3.94
Pulwonate snail| 5.63 | 6 | 4.95-6.13] 5.44 | 6 | 4.90-6.05 = = = 1.99 | 6 | 1.56-2.46 = = =

“Not measured.


ds

Appendix Table 4.

AND AMPHIPOD CHRONIC EXPOSURES

TOTAL SULFITE CONCENTRATIONS IN TEST CHAMBERS DURING FATHEAD MINNOW

(micrograms/liter)
Nominal “|
percentage Fathead minnow test hipod test
sewage
concentration Mean N Range Mean N Range
20 104 + 165% 53 nm? — 700 172 + 163 10 nm — 400
10 95 + 146 48 nm — 600 123 + 51 6 60 - 200
5 35 + 61 39 nm - 300 40 + 36 6 nm — 80
2.5 nm 32 nm — 200 nm 4 mm — 20
1.2 nm 32 nm - 60 nm 4 nm
} nn 33 nm nm 4 nm
(Control)

"Mean +1 standard deviation.

b

Not measurable, less than 20 ug/1.


FIGURES
Sewage Flow from Storage Tanks to Diluter Cells
Chronic Test Apparatus Used for Volumetrically
Dividing, Chlorinating, and Dechlorinating the
Sewage Effluent

Ozone Generator

Apparatus Used for Volumetrically Dividing and
Ozonating the Sewage Effluent in Chronic Tests

vi

1

12


TT

ve"on

Dry air flw —y

Py Air and 0;

oo rn 19" 3"
Aluminum foil 3/8" hole
round in stainless
in ante oteet tubing steal tubing
tubing
—— = ee,
—» --4 jl Y——> ——
————pr _/S

Figure 3.

Ozone generator.


occurred less readily, parthenogenesis was increased, and large numbers of
cleaving eggs had abnormal nuclei. In an early study Hubbs (1930)
concluded that the effects of ozone were cumulative and that concentrations
as low as 0.03 mg/1. were possibly lethal to minnows. In our study
lethal concentrations of ozonated effluent to fathead minnows were at
residuals approximately ten times higher than the concentrations

reported by Hubbs. Nightingale (1931) and Nebel et al. (1973) have
reported much higher fish survival in ozonated than in nondisinfected
effluents. Insufficient information is available at the present time for
suggesting criteria to protect aquatic life in natural water reaches
immediately adjacent to ozonation facilities.

Recommended criteria for chlorine have been proposed by Brungs (1973).

He concluded that for wastes treated continuously with chlorine the

more resistant organisms would be protected at residual chlorine
concentrations not exceeding 0.01 mg/1. and that concentrations not
exceeding 0.002 mg/1. would protect most aquatic life. Collins and
Deaner (1973) stated that dechlorination equipment should provide chlorine
concentrations in the final effluent of 0.1 mg/1. or less in accordance
with recent California requirements. Our results indicate that
continuous neutralization of the residual chlorine is necessary to
prevent harmful effects on sensitive organisms in the receiving water.

48


Appendix Table 2. CHEMICAL CHARACTERISTICS OF TEST-CHAMBER

WATERS DURING THE AMPHIPOD CHRONIC TEST

Nominal
percentage Nondisinfected Dechlortoated Chtortaated® Ozonated
En PP
a
20 7.30 | 5 | 7.11-7.45 | 7.29 | 4 | 7.097.368] 7.43] 4 |7.32-7.58 | 7.212 | 6 | 7.26-7.46
10 7.47 | 6 | 7,35-7.72 | 7.39 | 6 | 7.37.63] 7.52] 6 ]7.45-7.63 | 7.36 | 6 | 7.22-7.56
5 7.53 | 4 | 7.46-7.00 | 7.67 | 4 | 7.62-7.79] 7.46] 4 |7.63-7.96 | 7.57 | 4 | 7.50-7.68
25 7.63 | 7 | 7.45-7.78 | 7.62 | 7 | 7.52-7.83| 7.60| 7 |7.0-7.76 | 7.60 | 7 | 7.40-7.82
Lz 7.70 | 5 | 7.66-7.82 | 7.69 | 5 | 7.5%-7.85| 7.70] 5 |7-54-7.85 | 7.58 | 5 | 7.48-7.67
° 7.69 | 7 | 7537.82 |7.70 | 7 |7.s0-7.83 | 7.67 | 7 |7.52-7.83 | 7.50 | 7 | 6.98-7.86
(control)
Dissolved oxygen _(an/1.)
20 68 [so | 4.09.2 [74 Jar | 6.08.9 43 | 7.79.7 | 78 | 42 | 6.2-9.6
a0 a3 [4 | 7.39.8 |8.6 |37 | 6.99.8 38 | 8.39.8 | 2.6 | 42} 7.69.7
5 a7 [a | r092 Jes [35 | 7210.2] 9.0 | 40 | 2.59.9 | 27 | a6} 79-9.
25 7 |39 | 7.49.9 [9.1 |37 | 9.30.2] 9.1 | 36 | 8.4-10.0 a | 82-9.
12 a9 |37 | @.o-20.2 |9.2 |39 | 8.5-10.6] 9.0 | 36 | 3.4-10.0 33 | 8.29.8
0 9.0 1a! aza02 9.3 beo | estos! 9.2 | 42 | a.s-10.0! 9.0 | a} 5.29.9
Control)
Total hardness (as CaC0a, mg/l.)
20 62.3 | 4 | 59.0-67.0 Joa. | 4 | s¢.067.0/ 57.0 | 4 | 55.0-60.0| 63.5 | 4| 57.0-70.0
10 50.0 | 5 | s4.0-60.0 |s9.0 | 5 | s1.o72.0| s4.0| 5 | 50.0-58.0| 60.2 | 5| 52.0-80.0
5 52.2 | 5 | 50,0-54.0 |53.2 | 5 | 49.0-55.0/ 52.4} 5 | 48.0-56.0] 53.2 | 5] 53.0-55.0
23 |50.5 | 7 | 45.0-54.0 |50.1 | 7 | 49.0-51.0] 50.6 | 7 | 48.0-55.0] 52.1 | 7] 49.0-55.0
2.2 |51.8 | 5 | 50.0-55.0 [51.2 | 5 | 49.0-55.0] s1.8 | 5 | 49.0-56.0| 53.2 | 5/ so.osa.n
° 50.7 | 7 | 45.0-60.0 |52.2 | 7 | 47.0-65.9} 7 | 46.0-68.0] 53.7 | 7] 49,0-65.0
(Contro1)
Total adkalintty (as Cac
20 37.8 | 5 | 53.0-70.0 {50.8 | 5 | 43.0-60.0| 50.0 | 5 | 48.0-55.0{ 57.1 | 5{ so.0-67.0
10 49.7 | 6 | 45.0-55.0 |47.2 | 6 | 44.0-50.0] 47.1] 6 | 45.0-50.0| so.s| 6| 45.0-60.0
5 46.4 | 5} 65.0-50.0 {47.0 | 5 | 44.0-53.0| 45.8] 5 | 44.0-68.0) 48.6] 5] 63.0-55.0
2.5 [45.2 | 7| 43.0-48.0}43.6 | 7] 43.0-50.0] 45.6} 7 | 62.0-52.0] 43.8 | 7} 43.5-50.0
1.2 |4a.8 | 5} 42.0-57.0 [45.6 | 5 | 43.0-67.0] 46.8] 5 | 45.0-49.0) 47.6 | 5] 43.0-52.0
° aa9 | 7{ 42,0-49.0 [46.2 | 7 | 42.0-47.0] 46.8] 7 | 41.0-47.0 46.4] 7} 41.0-47.0
20 62] 5] 5.273] 67] 5] soss| 42] 5] ass] sc] 6| 44-75
0 aa] 6] s040] 45 | ¢] 405.46] 3.2] 6] 2.04.0] a4] 6f 3.062
5 2e| 5] 2533] 29 | 5] 2a62] 28] 5 3.2] 5} 3.03.3
25 27 | 7) 203.3] 28] 7] 2042] 26] 7] 528] 2.7] 7 a.736
12 as| s| 2.330] 28] 5] 233.9] 26] 5{ 2535] 2.7] 5| 2.33.3
fcutreay | 22] Tf BOSL| RE] 7] 2asol 27] 7! 2339] a5] 7 2370

“Wominel percentage sevage concentrations were 10, 5, 2.5, 1.2, 0.6, and 0, respectively.

"wedian vaiues.


82

Table 8. ACUTE TOXICITY OF CHLORINATED EFFLUENT TO SEVEN FISH AND SIX INVERTEBRATE SPECIES

Test Total residual chlorine TL50 concentrations (in g/l.)
temperature
Test species +#1°C Asher | 12-hr | %-hr = [48-hr | 72-hr | 96-hr | 5-day | 6-day |7-day
Fish
Brook trout a1 _ 360 200 175 175 135 130 93 83
Coho salmon 14 _ 230 230 105 102 102 95 95 83
Fathead minnow-Test 1 17 — 335 145 145 140 130 130 125 115
Fathead minnow-Test 2 18 >790 185 140 88 86 86 84 82 82
White sucker 16 >560 245 148 140 138 138 138 132 132
Walleye 12 _ 267 220 173 150 150 150 150 150
Yellow perch v7 >850 365 245 205 205 205 205 205 205
Largemouth bass a7 >574 494 345 310 300 295 272 261 261
Invertebrates

Amphipod-Test 1 17 _— _ 900 630 470 330 250 220 210
Amphipod~Test 2 18 _— | >810 | >810 660 255 215 185 177 t
Stonefly 18 >780 >780 >780 >780 480 400 270 235 195
Caddisfly 18 >740 >740 >740 >740 >740 >740 >740 >740 >550
Crayfish® 7 __ | >780 | >780 |>780 | >780 | >780 | >780 | >780 780
Operculate snail¢ 18 __ | >810 | >810 |{>810 | >a1o | >s1o | >s10 | >810 »
Pulmonate snail 18 _ | >810 | >a10 |>810 | >e10 | >810 | >810 | >810 | >810

"Mortality levels not recorded.
survival of controls became less than 90%.

Teste with crayfish and operculate snails extended through 12 and 14 days, respectively.


Sodium analyses (used as a tracer for determining diluter accuracy)

were conducted during 7 of 13 acute tests. These analyses showed that
all of the chlorinated and dechlorinated test concentrations and the
highest three ozonated and two highest nondisinfected test concentrations
were delivered within 2% of the nominal concentration. The remaining
experimental nondisinfected and ozonated concentrations were within

5% and 10%, respectively.

Acutely lethal concentrations of SO) were established by additions of
sulfur dioxide gas directly to the sewage effluent without prior
chlorination, but no toxicity was found after neutralization of sewage
chlorinated to high levels (16 mg/1.) followed by sulfur dioxide gas
neutralization. The amphipods were about five times more sensitive to

the measured residual sulfite than the fathead minnows. The 7-day TL50
values for fathead minnows and amphipods were 67 and 10 mg/1., respectively.
The dissolved oxygen concentrations bracketing these TL50 values for
fathead minnows and amphipods were 2.1-4.3 and 5.7-6.0 mg/1., respectively.
The bracketing pH levels were 6.3-6.8 and 6.5-7.7 mg/l. Thus suppressed
dissolved oxygen and decreased pH in the test waters may have contributed
to the toxicity.

Residual ozone concentrations between 0.2 and 0.3 mg/l. were lethal

to the fathead minnows. All deaths occurred in the first 1-3 hr of the
test. One test was continued for 96 hr, the.other for 7 days. The
measured dissolved oxygen in the test cells where the deaths occurred
had a supersaturation level of 120-125%. However, the next lower
concentration had a similar dissolved oxygen supersaturation level
without loss of fish.

29


SECTION VII

DISCUSSION

Disinfection with chlorine and ozone effectively reduced coliform
bacterial levels by 99.9%. After retention, the levels of total and
fecal coliforms were usually below 1,000 and 100 per 100 ml, respectively.
The disinfected effluents delivered to the diluters for the bioassays

had respective mean residual chlorine, sulfite, and ozone levels of
1.0-2.1, 0.4-4.3, and 0.0-0.5 mg/l. Residual ozone rapidly disappeared
from the effluent since residual concentrations of 1.0-2.0 mg/1. were
measured from the bottom of the ozone contact column, Shuval et al,
(1973) found a marked regrowth of coliform bacteria in both chlorinated
and dechlorinated wastewaters. In our study coliform regrowth was not
clearly demonstrated, probably because of the short retention times employed.

Only the chlorinated effluent consistently showed damaging effects to all
test species, The 7-day TL50 concentrations of total residual chlorine
ranged from 0.08 to >0.81 mg/l. for the test animals. Fish were more
sensitive than the invertebrate species during short-term exposures to
chlorine. These results agree closely with Brungs' (1973) conclusion that
salmonids are the most acutely sensitive to chlorine; next most sensitive
+ are warmwater fish and invertebrate species. The residual chlorine TL50
values for fathead minnows and amphipods agree closely with those given
by Arthur and Eaton (1971). Also, the acute toxicity of residual chlorine
for fathead minnows parallels that reported by the Michigan Department

of Natural Resources (1971) and Zillich (1972). The residual chlorine
96-hr TL50 for rainbow trout by the Michigan Department of Natural

44


Table 2. CHEMICAL CHARACTERISTICS OF THE SEWAGE

EFFLUENT IN THE LABORATORY STORAGE TANKS

Item Mean N Range
Temperature (°C) 14.2 87 9.0-20.6
pH 7.23 95 6.94-7.59
Dissolved oxygen (mg/1.) 4.7 91 0.9-8.8
Total hardness (mg/1.) 124 95 74-190
Total alkalinity (mg/1.) 131 96 90-177
Total acidity (mg/1.) 16.7 95 3.1-36.3
Ammonia nitrogen (mg/1.) 6.9 49 2.3-13.5
C.0.D. (mg/1.) 71.1 126 25.8-141.4
Sodium (mg/1.) 17.8 31 14.3-23.0
Total coliforms (per 100 m1) 676,000 144 20,000-10,100,000
Fecal coliforms (per 100 ml) 57,800 69 700-620 ,000

2, median value.

17


that all fish survived 96-hr tests when the concentration of NH3-N averaged
6.0 mg/1. and the MBAS concentration was 2.0 mg/l. Fifty per cent of the
fish died when NH3-N and MBAS concentrations reached 19.4 and 4.8 mg/l.,

respectively.

Ammonia nitrogen concentrations in our effluent from the storage tanks
averaged 6.9 mg/1. Fish deaths in some of the acute nondisinfected tests
were ascribed to low dissolved oxygen levels, but concentrations of

MBAS were not measured.

Dunstan and Menzel (1971) reported that secondary-treated sewage provided
an excellent source for continuously culturing and maintaining marine
Phytoplankton. Reproductive responses in the nondisinfected effluent by
both fish and invertebrates were generally similar to that of the controls.
This sewage source neither stimulated nor suppressed their survival and

fecundity,

No measurable toxicity to aquatic life was found from either short- or
long-term exposure to the ozonated effluent. Acute toxicity was only
demonstrable when special procedures were used to shorten the retention
time so that residual ozone could be maintained in the effluent
continuously. With these procedures residual ozone concentrations of
0.2-0.3 mg/1. were lethal to fathead minnows after 1-3 hours’ exposure.
By comparison, 0.19-0.34 mg/l. of residual chlorine was lethal to fathead
minnows after 12 hours' exposure. Although the toxicity magnitude for
these two disinfectants appeared to be alike, residual ozone had the
advantage of rapid dissipation from the effluent.

Little has been published on the toxicity of ozone to aquatic life,

particularly in wastewaters. MacLean et al. (1973) reported effects
of ozonated seawater on oyster eggs. They found that fertilization

47


9S

Appendix Table 3. TOTAL RESIDUAL CHLORINE CONCENTRATIONS IN TEST CHAMBERS

DURING FATHEAD MINNOW, AMPHIPOD, AND DAPHNID CHRONIC EXPOSURES

(micrograms/liter)
eae
|
‘peromntage: Mean Hoan Hesn_ x Range peire.
erase t +
sengeattnton 4
a | seasat 103 485 see | 7] w-a
20 no ~ mae
» | ases me] ow | 2-420 wesn | 6 | a06- us
‘ en ssa | 7 | a= s00 sew | s} ven
s a . so
a | wea se] a | on-am ssw] s| a-o
we esis | 39 | an- 96 wen | 5] om-2s
as u a 4
ne ar | oe | mos was [os] oem
a | esi | 10 | oan as ers | as | mew sea | o| mee
aa ‘ ‘ ‘
> | oss | mou cai} x | ooo asa |e] one
a | ase | se | ma re ee ee ter { s] mos
0.6 3 a 2
+ n | mos 1e2.| 0 | m6 ase | s| me?
. "
°
onteol) 2 o o | os
Amphipod test Teta
a favge | x | wae asi] 6 | 106~ 10 ane | s | s7-a0
1 a a3 336
af apen | a7 | 10-205 was | 5 | ur~ae uve | 6] aan
a | sons | a | 4 -a06 wen] 6 | ase asu | s| s-6
s “ 6 ey
» | sex | a | ae neo| s| mn vem] sf u-e
a | wen | a | moss wes] 0 | 7-35 aes [os] men
2s ry y 7
» | wea] oe | a-a0 ses] 6] usa nen |u] mo»
a * sae] 7 ze2 | | ams
2 Rn 4 2
» 2 2 sez] oo] oer zea | 6| me?
a {ess | ow | moss zas| ao] on? mei | s| mee
06 . 2 -
a [ores | oo | mea rez] ul ome mei | 6] one
a = = =
°
Seensrot)_ = =

Steen 1 atandard deviation.
Maat eeanerable, tase chen 1 u/s


DISCLAIMER

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

endorsement or recomméndation for use.

ii


The dechlorination apparatus failed on two occasions during the fathead
minnow and amphipod chronic tests (on July 8-9 and September 5-6, 1971)
and on October 18 during the second Daphnia test. During these times
residual chlorine concentrations of 0.2-1.1 mg/1. were measured in the
dechlorinated effluent. During the September failure 0.05-0.15 mg/1.
residual chlorine was found in the highest dechlorinated test
concentration, No failures were recorded with the dechlorination
apparatus used for the acute tests.

Analysis of sodium in the adult test-tank waters showed that the mean
sewage concentrations actually delivered were generally within 5 and 2.5%
of the nominal levels set for the respective fathead minnow and amphipod
chronic studies. Individual determinations overlapped the next
corresponding nominal test level about 15% of the time in the two highest
ozonated effluent concentrations. No similar overlap occurred with the

other three effluent treatments.

Fathead Minnow Chronic Test

More fathead minnows died in dilutions of chlorinated effluent than in
the other effluent treatments (appendix Table 5). Most deaths occurred
during the first 10 weeks of the test. During this time minnow survival
was significantly lower (P<0.05) in the two highest chlorinated
concentrations (> 42 ug/1. mean total residual chlorine). Survival in

these two concentrations was also lower at test termination.

No spawning occurred in the highest chlorinated effluent concentration
(110 ug/1.) (Table 10). No differences were observed in the spawning
results at the other test concentrations. Six deformed females were
found at test termination. These deformed females were not limited to
any one effluent treatment or test concentration, but were included in
the spawning calculations. The low egg hatchability in chlorinated

36


Starter and powdered dry grass and cereal leaves (Cerophyl) prepared
according to procedures of Biesinger and Christensen (1972).
Fleischmann's active dry yeast (7-8 g) was used for the October test.
Each food mixture was suspended in 9 1. of lake water. Performance of
the pump in delivering the food into the diluent test water was monitored
daily, and calculated respective amounts of food present were 1.7 + 0.6
and 1.4 + 0.7 mg/l. per day. Reproduction (parthenogenetic) occurred in
the test chambers after 1 week with a production of one or two generations
by termination. At test completion all test-chamber waters were
separately siphoned through a 60-wire-mesh stainless steel basket to
collect the daphnids for preservation in 70% ethanol. All daphnids

were counted in subsamples in petri dishes subdivided into l-cm? grids;

uniform microscope counting procedures were used.

Routine chemical analyses, in addition to those used for the acute tests,
were performed on the chronic test-chamber water (American Public

Health Association, 1971). For the fathead minnow and amphipod tests
dissolved oxygen analyses were usually conducted every other day; pH,
total hardness, total alkalinity, and acidity generally were measured twice
weekly; and water temperatures were recorded daily on two test chambers
in each effluent treatment. Sodium determinations were generally made
weekly with the flame photometric procedure until mid-November and
thereafter with the atomic absorption procedure. For the Daphnia tests
the only routine analyses conducted were for dissolved oxygen and pH;
they were done weekly on six test waters in each effluent treatment.

Samples for determining total residual chlorine, ozone, and sulfite

were collected from the test waters; protocol as described for the

acute tests was followed. Total residual chlorine analyses were conducted
daily during the workweek. The total residual sulfite procedure was not
developed until after the 14th week for the fathead minnow and” amphipod
tests, and then sulfite was measured weekly. As in the acute tests, residual

ozone was not measurable in any of the waters of the chronic tests.

34


Appendix Table 6.

SURVIVAL AND GROWTH OF SECOND GENERATION FATHEAD

MINNOW LARVAE AFTER 30 DAYS' CONTINUOUS EXPOSURE TO NONDISINFECTED

AND DISINFECTED SEWAGE EFFLUENT

Houta
percentage
‘eevee
emcantrattion
‘
20
a
4
10
2
a
s
ry
a
2s
2
a
2
2
a
0
(Control) B
a
»
”
a
3
.
a
as
a
a
12
a
a
0.6
s
ry
°
(Contre) 8

Final wuevts
‘Test eriala
rTzrs

87.5 | 100.0
1s
sof | _
soo ff
woo | 975]
90.0 | sso}
mo | 90.0] 45.0
cd ed
ons | ms}
ms | so]
as | 40.0
as tool
Borseae
ane
so] 00] 0.0
oof Lf _
ans | oams{
aso | 2s] ans
as} of _
as | s.0] 925
soo | ons} __
ys.o | szs]
aso} 225
as | sso}

Howtaal
oy Pina} percentare Mean
percentage ‘mean ‘eevage percentane

vetghe (g) || concentratton | Pinal percentage surviving | "surviving
Teak erials
ps zy
a 85.0
65.0 0.05 20
3 —- fe
a | 40 | ons | _
ns 0.07 10 wy
8 ss.0 | 60.0 | _
a wo} |
2 0.03 3 38.3
a | 6.0 | soo | _
a | aso} 9.0 |
664 0.08 2s 68.5
a {ans | eo. 0
a | 900 | 95.0 | _
60.1 0.05 88.3
a | wo f |
a] sas | ans |
50.5 0.05 °
(outros) 2 | 45.0 | 97.5 | 42.5
> Ononated
& | 95.0 | 95.0 1 90.0
0.0 _ 20 99.0
atoms | 0]
Lz 0.02 20 7.8
2 ms | 9.0] _
Sra | ozs fans |
26.0 0.08 s 35.0
3 ro | 1.0 | _
a woo}
76.9 o.0s oar
2 =
a | 38.0 | 10.0 f
20.0 0.05 Ted
a | es] ss0 f _
60.0 | a.0 | 10.0
52.0 0.08 ° 49.2
(oarron » | 45.0 | 35.0 | 45.0

0.05

0.05

“Test erial vas not conducted.

"mean total residusl chlorine levels for the six test concentrations (io ug/l.) ware 198, 88, 21,

“Lorvas ware taken from control tanks.

59

1, and nonseasurable, respectively.


SECTION III

INTRODUCTION

Federal and most state pollution control regulations require disinfection
of sewage effluents prior to discharge. By 1977 treatment of all
municipal effluents will be required to achieve a fecal coliform level of
less than 200 per 100 ml (U. S. Environmental Protection Agency, 1973).
Historically, chlorination has been a widely practiced means of disinfection
in this country. However, recent concern over the toxicity of chlorinated
effluents to aquatic life has stimulated a re-evaluation of effluent-
disinfection practices. The toxicity of chlorine to aquatic life in
laboratory and field situations has been summarized recently by Brungs
(1973). Besides the effects of chlorination, municipal wastewater
toxicity has also been attributed by Esvelt et al. (1973) and Tsai (1973)
to other components such as methylene blue active substances (MBAS),
ammonia, and turbidity.

Ozonation and chlorination followed by dechlorination are alternative
methods for disinfecting wastewaters. By means of short-term exposures
Zillich (1972) and Esvelt et al. (1973) compared the toxicity of
chlorinated and dechlorinated wastewaters to fish. In an earlier study
Kelly et al. (1960) showed that the feeding activity of oysters was
suppressed in both chlorinated and dechlorinated water but not in untreated
or ultraviolet-sterilized water. Ozonation is being widely studied and
‘appears to offer many advantages over the use of chlorine (Sliter, 1974).
Although the effects of ozonating natural waters have been reported


highest dechlorinated concentration (100%) was always less than 50% for
the 13 species tested. Similarly, no long-term effects were noted on the
three species tested in the highest dechlorinated concentration (20%),
except those ascribed to failure of the dechlorinator. Residual sulfite
concentrations ranged from <20 to 700 ug/1. (as S02) in this chronic test
dilution. In addition, no fathead minnows died in the 7-day tests when
chlorination of the effluent to very high levels was followed by
neutralization with sulfur dioxide.

Other studies have also shown that chlorinated effluents are more toxic
than effluents that have been dechlorinated after chlorination. Onsite
tests by Zillich (1972) at two Michigan wastewater plants showed that
thiosulfate dechlorination effectively removed the toxicity from
chlorination. As we found in our study, he also found that dechlorinator
failure resulted in acute chlorine toxicity. Esvelt et al. (1973) have
shown that dechlorination of San Francisco wastewater with bisulfite also
removed the toxicity from chlorination. Collins and Deaner (1973) have
advocated sulfur dioxide as an inexpensive and effective means for
neutralizing chlorinated effluent toxicity.

Acute toxicity was observed when the effluent was purposely dosed with
elevated amounts of sulfur dioxide. The 7-day TL50 values for amphipods
and fathead minnows ranged from 10 to 60 mg/l. (as sulfite). At these
lethal levels the dissolved oxygen concentrations and pH were suppressed.
A principal disadvantage of sulfite dechlorination is the need to balance
dosages carefully to prevent toxic excesses of either chlorine or sulfite.

Municipal wastewater toxicity is not limited to chlorine. Esvelt et al.
(1973) have attributed the toxicity of municipal wastewaters to MBAS
and ammonia nitrogen (NH3-N) as well as to chlorination. Using an

effluent from an oxidation pond receiving primary effluent, they found

46


3.

SECTION IX

APPENDICES

Chemical Characteristics of Test-Chamber Waters
During the Fathead Minnow Chronic Test

Chemical Characteristics of Test-Chamber Waters
During the Amphipod Chronic Test

Total Residual Chlorine Concentrations in Test
Chambers During Fathead Minnow, Amphipod, and
Daphnid Chronic Exposures

Total Sulfite Concentrations in Test Chambers
During Fathead Minnow and Amphipod Chronic
Exposures

Number of Surviving Fathead Minnows after 10, 25, and
43 Weeks' Continuous Exposure to Nondisinfected and
Disinfected Sewage Effluent

Survival and Growth of Second Generation Fathead

Minnow Larvae after 30 Days’ Continuous Exposure
to Nondisinfected and Disinfected Sewage Effluent

52

55

56

57

58

59


SECTION IV

GENERAL BIOASSAY AND ANALYTICAL PROCEDURES

The secondary sewage treatment plant of Silver Bay, Minnesota, on the
north shore of Lake Superior (population 3,500) was chosen as the effluent
source. The municipality's water supply is taken directly from Lake
Superior, as is the water supply for the National Water Quality
Laboratory. Silver Bay's secondary sewage treatment plant employs a
trickling filter. The effluent disinfection treatments used in this
study were designed to approximate current field conditions.

DISINFECTION

Effluent was collected by pump from the treatment plant's final settling
basin before chlorination and was transported by truck to the National
Water Quality Laboratory, a distance of 80 km. The three cylindrical
steel tanks on the truck had a combined capacity for hauling 4,550 1.

Upon arrival at the laboratory the sewage effluent was pumped into three
1.8-m-diameter elevated tanks, each with a storage capacity of 1,900 1.
The stored sewage was kept at about 15° C by recirculating chilled lake
water through stainless steel coils in the bottom of the storage tanks.
The general effluent flow design for disinfection is given in Figure l.
From the storage tanks sewage was pumped into two stainless steel head
boxes for distribution and for the disinfection steps before acute and
chronic bioassay. When acute and chronic tests were conducted simultaneously,
3,200-3,800 1. per day of effluent were needed. Between 1,100 and 1,700 1.
per day were used for the fathead minnow and amphipod chronic tests.


EPA-600/3-75-012
November 1975

COMPARATIVE TOXICITY OF SEWAGE-EFFLUENT DISINFECTION TO

FRESHWATER AQBAPER LIFE

by

John W. Arthur
Robert W. Andrew
Vincent R. Mattson
Donald T. Olson
Gary E. Glass
Barbara J. Halligan
Charles T. Walbridge

Environmental Research Laboratory
Duluth, Minnesota 55804

U.S. ENVIRONMENTAL PROTECTION AGENCY

OFFICE OF RESEARCH AND DEVELOPMENT

ENVIRONMENTAL RESEARCH LABORATORY
DULUTH, MINNESOTA 55804


effluent concentration more than 50% of the test animals died in 2 of
12 tests, and more than 20% died in 6 of 12 tests. This higher mortality
in the nondisinfected effluent may have been related to reduced dissolved

oxygen concentrations in these test waters.

Progressively higher concentrations of effluent delivered to the test
tanks reduced the dissolved oxygen concentration. The lowest dissolved
oxygen concentrations were found in dilutions of the nondisinfected
effluent. For any individual test the mean dissolved oxygen concentrations
were above 3.0 mg/l. (about 30% saturation). Occasional measurements
below this level were recorded in the nondisinfected tests with brook
trout, coho salmon, sucker, amphipod (test 1), and stonefly. Mean
dissolved oxygen measurements less than 50% saturation were typically
found in the three highest concentrations of nondisinfected effluent
and the two highest levels of dechlorinated effluent. Mean dissolved
oxygen concentrations were consistently above 50% saturation in the
ozonated and chlorinated test waters. The range in pH for all the test-

chamber waters was from 6.9 to 7.9 units.

The chlorinated effluent was more acutely toxic to fish than to
invertebrates. The 7-day TL50 concentrations of total residual chlorine
for fish and invertebrates ranged from 0.08 to 0.26 and 0.21 to >0.81 mg/1.,
respectively (Table 8). The mean total residual chlorine concentrations
throughout the entire test were used for calculating the TL50 values.
Brook trout and coho salmon were the most sensitive fish species, and
amphipods the most sensitive invertebrate. Flow-through acute tests were
not done with Daphnia magna, Fish died more quickly than invertebrates
in the chlorinated effluent. Most of the fish died in the first 24 hr of
the test, but 24-48 hr were needed to kill 50% of the amphipods, 72 hr
were needed for the stoneflies, and more than 6 days for the remaining
invertebrate species tested. Fifty percent of the operculate snails
(Campeloma decisium) died only after 14 days' exposure to the effluent.

27


Sodium levels were measured to determine the concentration of effluent
actually delivered to the acute and chronic test tanks. A flame
photometric method was initially used. Later, sodium concentrations were
determined by direct aspiration of the aqueous samples into a Perkin-
Elmer 403 atomic absorption spectrometer (Perkin-Elmer Corporation, 1971).

RESULTS

The modified amperometric methods developed for this study were more
sensitive than procedures given by the American Public Health Association
(1971). Fifty-milliliter samples of measured concentrations of tap
water chloramines diluted serially five~ to a hundredfold with distilled
water gave recoveries of 77-107%. This corresponded to a mean standard
error of approximately 15% in the range of 10-200 ug/l. total residual
chlorine. A thousandfold dilution, or approximately 1 ug/l., gave
recoveries of + 100% corresponding to the lower limit of detection.
Difficulties in measuring samples <10 ug/l. were partly due to inaccuracies
in delivering + 0.01 ml of titrant. For the sulfite measurements,
titrations were reproducible and sensitive to 20 ug/l.

The effluent was not uniform and required daily monitoring. The

chemical characteristics of the effluent are shown in Tables 1 and 2.

In the treatment-plant analyses the greatest variations were found in

the measurements of solids and B.0.D. The largest variations in the
laboratory storage tanks were in total and fecal coliforms, C.0.D., acidity,
and dissolved oxygen. The ammonia nitrogen concentrations analyzed from
the diluters were similar, and all mean values were within 1 mg/l. of

the mean storage tank concentration.
The concentrations of residual chlorine, ozone, and sulfite maintained

in the effluent after disinfection are listed in Table 3. Total
residual chlorine and ozone analyses were done daily, and sulfite cell

15


RESEARCH REPORTING SERIES

Research reports of the Office of Research and Development,

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

1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research

4. Environmental Monitoring

5. Socioeconomic Environmental Studies

This report has been assigned to the ECOLOGICAL RESEARCH series.
This series describes research on the effects of pollution on
humans, plant and animal species, and materials. Problems are
assessed for their long- and short-term influences. Investiga-—
tions include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work
provides the technical basis for setting standards to minimize
undesirable changes in living organisms in the aquatic, terres—
trial and atmospheric environments.

This document is available to the public through the National
Technical Information Service, Springfield, Virginia 22161.


chamber 5B may be due to factors other than residual chlorine. For

unknown reasons spawning by the minnows was not confined to the undersurface
of the concrete tiles. Thirty-six per cent of the spawning occurred on

the egg cup undersurfaces and on two or three occasions on standpipe

screens.

Overall egg hatchability was lower in the highest dechlorinated
concentration (Table 10). For comparison, seven groups of eggs spawned
on different days in one of the highest dechlorinated test chambers
were incubated in a control chamber. The average hatchability was 87%
(range 78-90%). By contrast, two groups of eggs from the control
chambers incubated in one of the highest dechlorinated chambers gave
hatchability values of 56% and 54%.

Survival data for the second generation of fathead minnow larvae after 30
days' exposure in progeny tanks are given in appendix Table 6. Two to five
trials were conducted at each effluent test concentration. Final

survival varied widely. The lowest survivals of minnow larvae occurred

in the two highest chlorinated concentrations containing mean total
residual chlorine levels of >88 ug/l. Survival was reduced in mean chlorine
levels of 21 ug/1., but the variation in final survival was mch greater
(2.5-47.5%) 6

Amphipod Chronic Test

Amphipod survival was noticeably lower after 10 weeks in chambers with the
highest chlorinated and dechlorinated effluent concentrations (appendix
Table 7). Poor survival in the highest dechlorinated concentration was
probably due to failures of the dechlorinator. Survival after 16 weeks
of the test was also lower in the second highest chlorinated concentration
(54 ug/l. mean total residual chlorine).

39


€@

Table 6. SOURCES AND SIZES OF ANIMALS USED FOR THE ACUTE TESTS

Mean
Test species Source length (cm)
Fish
Brook trout National Water Quality Laboratory, Duluth, Minnesota 11.0
Coho salmon Western Fish Toxicology Station, Corvallis, Oregon 8.7
Fathead minnow National Water Quality Laboratory, Duluth, Minnesota 3.1
White sucker State Fish Hatchery, Grand Rapids, Minnesota 6.7
” Walleye State Fish Hatchery, Aitken, Minnesota 6.1
Yellow perch Park Lake, Mahtowa, Minnesota 4.9
Largemouth bass Federal Fish Hatchery, New London, Minnesota 7.0
Invertebrates
Amphipods Eau Claire River, Gordon, Wisconsin Adult stage
(Gammarus pseudolimaeus)
Stonefly Blackhoof River, Carlton County, Minnesota Nymph stage
(Pteronarcys sp)
Caddisfly Pequaywan Lake Outlet, St. Louis County, Minnesota Nymph stage
(Hydropayche sp)
Crayfish Eau Claire River, Gordon, Wisconsin 2.8
(Orconectes virilis)
Operculate snail St. Croix Riyer, Gordon, Wisconsin 1.6
(Campeloma decisum)
Pulmonate snail Zau Claire River, Gordon, Wisconsin 0.7
(@hysa inteera)



SECTION VI

CHRONIC TESTS
METHODS

Modified proportional diluters served as the water-delivery system, and
the diluent source was raw Lake Superior water. The nominal sewage
concentrations used for tests of the nondisinfected, dechlorinated, and
ozonated effluent were 20, 10, 5, 2.5, and 1.2%. For the chlorinated
effluent, the nominal sewage concentrations were set at one-half of the
other three treatments. Duplicate test chambers were used for each
experimental test concentration and for the controls. The diluters
cycled at approximately 5-min intervals throughout the tests. Flow-
splitting cells divided each test concentration at a 2-to-1 ratio for
delivery to adult and progeny test chambers. Durotest (Optima FS) and
widespectrum Gro-lux fluorescent bulbs provided the light source for the
chronic tests.

The fathead minnow chronic test was started on June 3, 1971. Thirty-

five hatched fry, 1-20 days old, were randomly assigned to each adult (27-1.)
tank. Fry were obtained from National Water Quality Laboratory cultures.
Mean test-chamber retention time was maintained at about 4 hr, and water
temperature was maintained at 24° + 2° C, Additional procedures for this test
followed the methods recommended by the National Water Quality Laboratory (1971).
The chambers were cleaned daily 1-2 hr after feeding, since excess food
temporarily lowered the residual chlorine test concentrations. ,The first
spawning was recorded on October 27. Hatched larvae were either discarded

or placed into corresponding progeny (14-1.) tanks. Spawning of the

minnows ceased on March 24, 1972, and the test terminated on April 1, 1972.

32


8T

Table 3. TOTAL RESIDUAL CHLORINE AND OZONE CONCENTRATIONS MEASURED FROM DILUTER TOXICANT-BEARING CELLS AND OZONE FROM COLUMN

DURING THE ACUTE AND CHRONIC TESTS
(oi.114grams/liter)

. Diluter toxtcant-bearing celle (in mg/l.)
ethotelidine method —| Ozone column
Test Residual guifite Residual chlorine Residual ozone Residual chlorine Residual ozone (mg/1,)_
organism {-Hean f ¥ f “Range Hean f xy ‘Range esi} w | ‘Range ‘Wean x Range ean } x | Range
Brook trout 1.35] 4 | 0,6-2.22 | 2.63] 8] 2,39-1.94 | 0.03] 5 | o.o1-0.04 |0.86 | @ | 0.71-0.99) _* | _ _
Coho salmon a on 1.74] 6| 1,482.03 | 0.04] 7 | 0.01-0.08 0.85 | 7 | 0.58-0.98; _ | __ _
Fathead’ minnow —}]—| — 1.26] 7| 0.39-2.73 | 0,06] 6 | 0.01-0.07 0.50 | 7 | 0.17-1.50{ _ | _ _
Fathead minnow or 1.99] 8] 1.692.285 | _ | _ 1.06 | 8 | 0.65-1.25{ _ | __ _
White sucker a oe 1.s2| 8} 0.70-2.30 | 0.06] 5 | 0,02-0.08 [0.85 | 7 | 070-105) _ | _ _
Walleye —~J—[ _ 1.98{ 7] 2.77-2.23 | 0.17] 7 | 0.05-0.30 Jo.g99 | 6 | oes-tas] _ | __ _
Yellow perch 0.90) 2 | 0.80.9 | 2.10] 8] 1.80-2.47 | 0.02) 8 |<o.001-0.10 |1.16 | 6 | 1.00-1.40] 1.95 | 4] 1.16-2.33
Largemouth bass | 1.30] 2 | 1.1-1.4 | 2.97] 8{ 1.61-2.26 | 0.02] 2 _ 1.28 | 7 | 1.06-1.48] 2.07 | 3] 2042.20
Invertebrates
Amphipod -|J- _ 1.77] 8| 0.03-2.60 | 0.02] 6 | <0.001-0.06 1.02 | 2 | 0.75—1.30} 2 | __ _
Amphipod ~f{oe}]oo 2.22] 8| 1.73-2.78 | 0.02] 6 |<0.001-0.02 1.55 | 2 | 1.55 2.32 | 6] 2.72-2.67
Stonefly 0.43 2 }0.38-0,48 | 1.73] 8] a.29-2.98 | 0.16] 7 |<0.001-0.62 |a.a5 | 5 | 1.20-1.27] 2.73 | 5] 1.05-2.66
Caddiafly a en 1.7%] 8] 1.42-2.03 | 0.01} 3 | 0.004-0.02 | _ |_ _ —~fe— _
Crayfieh —~Joe} o. 19a] ar) 1.80-2.27 | _ | _ _ aa6 [12 | 0.80-1.35} _ | _
Operculate snatt | | | __ 2.05| 13] 1.69-2.75 | 0,01] 10 |<0.001-0.06 1.24 | 9 | 0.65-1.55| 2.20 | 9] 1.72-2.71
Fotmonace email | 7 of 2,22) 6] 1,732.78 | 0.02) 6 |<0.001-0.18 |2.55 | 2 | 1.55 2,32 \ 6} 1.72-2.67
4,30 | 25 | 0.1-24.3 | 2.30 |332 | <0.001-3.61 <0,001-0,17 } soz hoe | 0.05.13! 1.03 [339 | <o.oo1-2.89
2.70] 6 | 0.2-5.2 2.16 ]146 | 0,643.93 | 0,01 ]113 |<0.002-0.11 | ° dy) . .
on en ee 2.26] 14] 1.64-2.71 | 0,47] 4 | <0.001-1.0 a _ 1.62 | 7] 0,50-2.37
ne 2.04 | a2 | 2.22-3,49 | 0.07] 7 |<o.001-0.32 | __ |__ _ 1.20 | 6} 0.45-2.87



8.

9.

Residual sulfite at the concentrations used to neutralize the
residual chlorine dissipated readily and had no adverse effect on
the species tested.

When special test procedures and shorter retention times for the
ozonated effluent were used, measured residual ozone was about

as lethal as residual chlorine to fathead minnows.

Amperometric procedures were developed and used for analyzing total
residual concentrations of chlorine, ozone, and sulfite. Total
residual chlorine concentrations as low as 10 yg/1. were readily
measurable with a lower limit of detection of 1 ug/l.


Appendix Table 1.

WATERS DURING THE FATHEAD MINNOW CHRONIC TEST

CHEMICAL CHARACTERISTICS OF TEST-CHAMBER

Nominal
percentage Nondisinfected Dechtorinaced Chlorinated” zona
le mS OP
ae.
20 7.32 | 12| 7.25-7.46 7.28 | 12 |7.00-7.30| 7.40 | 12 | 7.26-7.60 | 7.16 [12] 6.83-7.50
20 7.45 | 12| 7.20-7.63) 7.36] 22 |7.05-7.55| 7.51 | 12 | 7.29-7.76 | 7.26 |12 | 6.95-7.50
3 7.56 | 32) 7.31-7.70) 7.47 | 12 |7.15-7.60 | 7.59 | 22 | 7.35-7.78 | 7.38 |22 | 7.06-7.62
25 7.61| 12 | 7.30-7.86 7.57} 13 |7.15-7.80| 7.61 | 13 | 7.29-7.83 | 7.41 1134 7.06-7.68
12 7.64 | 12] 7.35-7.88) 7.66 | 12 |7.49-7.85 | 7.67 | 12 | 7.50-7.85 | 7.43 |22 | 7.05-7.64
° 7.66 | 12 ea 7.69 | 12 |7.50-7.86 | 7.68 | 12 | 7.46-7.86 | 7.46 ]22 | 7.25-7.75
(Control)
Dissolved
20 SL | MH) 2574] 5.3 169 1 3.074 16.6 | 6B] 4.98.2 | 5.5 [67] 2.97.5
10 6.2 | se| 3.97.6] 60 | 65 | 2.9-7.9 | 69 | 66] 4.08.0 | 6.5 Joa] 42-82
5 6.4 | 68| 4.7-7.8] 6.6 | 59 | 4.3-7.9 | 7.0 | 66| 61-80 | 6.9 [60] 4.98.6
25 6.8 | 62| 4.07.7] 7.2 | 55 | 6.67.9 | 7.2 | 62] 6.682 | 7.0 jer} 5.38.6
12 7.0 | 60] 5.80.0] 7.2 | 59 | 6.082 | 7.2 | 66] 6.48.1 | 7.0 Jer) 5.78.5
coer | &2 12] Hee0] 72 [oe | so02 [72 [or] 5.280 | 69 Jos] s.r0s
Fotal hardoess (as Ca0o:
20 60.8 | 11 | 54.0-70.0| 59.8 | 11 |53.0-66.0 [59.9 | 22 | 53.0-66.0 [60.2 [x1 | 53.0-72.0
20 34.7 | 2 | 47.0-64.0| 55.7 | 11 |52.0-70.0 |53.4 | 11 | 48.0-60.0 |53.5 {22 | 49.0-57.0
5 51.6 | 12 | 68,0-56.0] 5i.8 | 12 |47.0-58.0 |52.1 | 32 | 48.0-56.0 [52.8 [12 | 48.0-57.0
2.5 |50.0 | 12 | 49.0-51.0| 49.8 | 12 |4s.0-54.0 |50.3 | 22 | 48.5-52.0 [50.0 [12 | 47.0-59.0
2.2 | 52.3 [a1 | 47.0-60.0| 50.6 | 11 |47.0-55.0 }51.2 | 31 | 47.0-53.0 |51.6 |10 | 47.0-60.0
° 49.3, | 12 | 47.0-50.0] 48.3 | 11 ]46.0-51.0 ]49.0 | 12 | 45.0-53.0 |49.8 [22 | 46.0-57.0
(Control)
Ikalinity (as C200,
20 33.4 | 12| 50.06: { 50.9 | 13 | 4s.0-61.0|52.6 | 12| 47.0-62.0|52.8 |12| 44.0-63.0
10 50.0 | 12] 46.0-54.4 47.9 | 12 | 45.0-53.0] 49.0 | 12} 46.0-59.0|48.6 |22| 45.0-58.0
5 47.3 | 12] 46.0-55.4 66.8 | 12 | 43.0-51.0/ 46.3 | 12} 42.0-53.0|46.6 |12| 44.0-51.0
2s [45.5 | 11] 43.0-52.q 45.2 | 12 | 43.0-50.0/ 45.6 | 12] 43.0-50.0]45.4 [12] 42.0-53.0
22 [44.9 | a2] s2.0-50.0 45.5 | 12 | 42.0-52.0] 46.7 | 21] 40.0-50.0]45.4 |21] 42.0-50.0
° 46.1 | 12| 62.0-50.9 45.7 | a1 | 42,0-50.0| 46.0 | 12] 42.0-50.0] 46.5 | 11] 44.0-53.0
(Control) .
tty (as/1.)
20 5.6 | 32 on 7.2 | 12 | s.on0| 5.9 | az| s.99 | 6.2 [12] 42-209
10 42 | a2] 2.05.9] 4.9 | a | 35-74 | 4.0 | a2] 2.85.0 | 4.8 | 22 6.9
5 39 | 2] 25-99] 4.2 | a2 | 289.9 | 35 | 2] 2.588 | 40 |12) 2.59.9
25 aa {um} asso} 3a] 12 | 235.9 | 30 | a2] i950 | 3.7 [22] 2.36.4
1.2 29 | a{ 235.0) 28] 1 | 234.0 | 29 | m{ 2.94.0 | 35 far] 2.56.5
o 3a] um} 2062] 29 | 11 | 2.05.6 | 2.9 | 20] 205.0 | 3.3 [11] 2.05.9
(Control)

“Wominal percentage sevage concentrations were 10, 5, 2.5, 1.

"vedien values.

54

, 0.6, and 0, respectively.


Reproduction was significantly reduced in the three highest chlorinated
effluent concentrations. Second generation young were not produced at
either the 123 or 54 ug/l. mean concentrations (Table 11). The
reproductive index was significantly lower in the 19 ug/l. chambers

as compared to the controls (P <0.05). The highest measured level of
residual chlorine having no effect on the amphipods was 12 yg/1.

Survival and growth of the second amphipod generation were similar after
30 days in all concentrations (appendix Table 8). The second generation
appeared to survive somewhat better overall (83.5%) than did the original
adults after 30 days (64.4%). Survival of both generations was
generally similar during the first 30 days of exposure.

Daphnia Chronic Test

Greater numbers of the Daphnia initially added to test chambers survived
to termination in the first of the two tests performed (Table 12).
Survival and reproduction of the daphnids were similar in the control
chambers with the two different food sources used. Elimination of the
daphnids during the first week of the second test at the highest
dechlorinated effluent concentration was probably related to failure

of the dechlorinator.

Daphnia did not survive the 2 weeks' exposure in the three highest
chlorinated effluent concentrations. In these three concentrations

death occurred during the first week of exposure, and no young were
recoverable at test termination. Increased daphnid numbers at 12 days in

a few of the disinfected and nondisinfected effluent concentrations were
due to counting errors. Calculation procedure used for determining young
per adult is given in Table 12, and the results were statistically analyzed.
The highest level of total residual chlorine having no significant

effect on Daphnia was 2-4 ug/l. (P <0.05).

40


Table 11 (continued).

SURVIVAL AND REPRODUCTION OF AMPHIPODS AFTER

20 WEEKS' CONTINUOUS EXPOSURE TO NONDISINFECTED
AND DISINFECTED EFFLUENTS

Nominal Total
Percentage Final Final number Young, Births
Sewage male/female | gravid young Total per per Reproductive?
seoncentration ratio females | produced | births | female | female index
Dechlorinated
A 2s 4 152 4 30.2 0.8 1.6
20
B C) 0 48 2 _ _ °
A 8/4 4 aad 8 85.2 2.0 3.0
10
B 7/4 3 422 1 105.5 2.8 3.5
A 8/3 3 283 8 94.3 | -2.7 3.7
5
B 3/6 5 487 u 81.2 1.8 27
A 4/uL 7 740 22 67.3 2.0 2.6
2.5
B 8/9 8 907 23 100.8 2.6 3.4
A 6/6 5 482 2B 80.3 2.2 3.0
1.2
B 8/6 6 501 16 83.5 27 3.7
A 8/6 5 290 Ww 48.3 2.0 2.8
0
(Control) 8B 914 4 183 7 45.8 1.8 2.8
Ozonated
A 15 3 e18 a 163.6 4.2 4.8
20
B 8/5 2 429 n 85.8 24 2.8
A 4/4 4 487 1 121.8 3.5 AS
10
Fy 6/5 1 722 20 144.4 4.0 4.2
A 4/7 4 a2 uv 63.1 2.4 3.0
5
B 75 3 478 et} 95.6 2.8 3.4
A 5/8 5 502 16 62.8 2.0 2.6
2.5
B 3/9 5 465 Wv 51.7 1.9 24
A 9/5 3 708 a 141.6 42 4.8
12
B 5/5 1 679 19 135.8 3.8 4.0
A 9/3 2 164 5 54.7 17 # 2.3
0
(Control) _B 5/8 5 46 2 5.8 0.25 0.9

“Determined by adding total births (spawnings) plus final gravid females divided by final number of
surviving females.

initial number of amphipods per test was 35, thinned to 15 individuals after 60 days.

Mean total residual chlorine levele for the six test concentrations (in ug/l.) were 123, 54, 19, 12, 8,

and nonmeasurable, respectively.

42


(Benoit and Matlin, 1966; Institute of Maritime Fisheries, 1972; MacLean
et al., 1973), very little has been done to determine the toxicity of
ozonated effluents to higher aquatic life.

Collins and Deaner (1973) compared the toxicity of water immediately
above and at points 100-300 ft downstream from a chlorinated wastewater
plume. All king salmon fry died within 14 hr in the downstream waters
(total chlorine residuals of 0.2-0.3 mg/1.), and none died in the
upstream water. When chlorination was temporarily shut off at four
wastewater treatment plants by the Michigan Department of Natural
Resources (1971), the survival of brook trout and fathead minnows in
cages below these plants was usually much higher.

The need for additional information on the toxicity of effluent
disinfectants to aquatic life led to the present investigation. The
purposes of this study were to compare with short- and long-term flow-
through bioassays the toxicity of a domestic sewage effluent after (1)
chlorination, (2) chlorination followed by dechlorination (hereafter
called dechlorination), and (3) ozonation. At the same time tests were
conducted with the nondisinfected effluent. The purpose of the short-
term tests was to determine the lethality of these four effluent
treatments to several fish and invertebrate species, The intent of the
long-term tests was to establish concentrations of nondisinfected and
disinfected effluents having no effect on three aquatic species over a
life cycle.

The municipal effluent used in this study had the following necessary
characteristics:
1) it had been subjected to secondary treatment;
2) chemical characteristics were typical of secondary municipal
effluents throughout the country;
3) it did not contain toxic industrial wastes; and
4) its diluent water was similar in quality to the test water.