UNITED STATES
ENVIRONMENTAL PROTECTION AGENCY
REGION I £SJ>
BIOMONITORING PROTOCOLS
JULY 1, 1990
Printed on Recycled Paper
-------
ATTACHMENT A
Example Chain of Custody Documentation
(July 1, 1990) 1
Printed on Recycled Paper
-------
ATTACHMENT A
Example Chain of Custody Documentation*
Chain of Custody Documentation provides vital sample information
and establishes a clear paper trail for tracking the sample from
the moment of collection to the final reporting of analytical
results. This subsequently upholds the integrity and quality of
the reported biomonitoring results.
Chain of Custody Tape to seal the container: The tape has a
sample number which corresponds to a Field Data Card (see below),
and provides collection date and time, inspector's/sampler's
signature, name and title information. If the tape is broken
prior to the onset of analysis, this may indicate tampering and
decrease sample integrity.
Sample Tag; The tag remains attached to the sample container and
provides the following: source of sample; date and time of
collection; preservation technique; analyses to be performed, a
sample number which corresponds to the Field Data Card (see
below); sampling crew identification and sample type (composite
vs. grab/chlorinated vs. prechlorinated). The tag should be
water resistent to improve longevity and may also be used to note
special remarks about the sample.
Field Data Card; This card basically replicates the information
contained on the sample tag and also records other environmental
observations and analytical field results made at the time of
collection. Additionally, the card is marked with a pre-printed
sample number which is used to trace back to the sample tag and
the actual sample in the laboratory's possession. The card is
kept with the project coordinator or individual who ultimately
receives the analytical report.
Chain of Custody Record; This form is completed by the sampling
crew and is submitted along with the tagged samples to the
analytical laboratory, after the sampler signs off and dates the
"Relinquished" line and a laboratory representative signs off and
dates the "Received" line. The Record should be duplicated so
that the receiving laboratory and the project coordinator/sampler
each have a copy of the signed document.
'These forms are provided as examples only.
(July 1, 1990) 3
Printed on Recycled Paper
-------
SAMPLE A
Field Data Card
SOURCE'
CITY
PWM.T N0
COLLECTOR
WEATHER g£JAfl>flAlN SNOW (CIRCLE ONE)
AIR TEMPCCl < O'^JJ^ii'H)1 >2i-
SAMPLE LOCATION SUPPLY WAT
(Circle On«|
LAB CODE \'u
__
"«OJECT ifj I I
STATION
SAMPLE
COLLECTION DATE
Y M M D D Y YM M 0 Q
RAW TREATED
START
COLLECTION TIME
Bteti
BOO
TSS
Turb
Organic!
VOAl
START e*D
SAMPLE TEMP CQ fflB
PM(SU)
TOTCIjjmg/l)
FLOW MEASUREMENT
None
Magnww
Vcniun
P«rsn«n
V Notcn
R«CI«ngular
Oin«r
MULTIPLIER XI
Chain of Custody Tape
farm
—• ttf^m on vi w »iri«k.k«iT «•>•«•
(July 1, 1990)
Sample Tag
J
-------
SAMPLE B
SOURCE
CITY
PERMIT N0
COLLECTOR
WEATHEjTgLEAg RAIN. SNOW (CIRCLE ONE)
AIR TEMP('C)
~ " ir— ^
SAMPLE LOCATION SUPPLY WATE
Organic s
VOAt
FLOW MEASUREMENT
None
Magmatar
Veniuri
Pannaii
V-Noicrt
Raclangular
LAB CODE W 55SC |
PROJECT tM I I I I i
STATION i
SAMPLE TYPE
COLLECTION DATE
Y M M D D . Y.YMMPD
END
START
COLLECTION TIME
QUL021
START END
SAMPLE TEMP CC)
TOTCI^mg/lj
ILE SOLIDS (mi/I)
FLOW METER
TOTALIZER
START
END
MULTIPLIFB Jf I
Tjpe
Sample so
i»n.-
firf
(July 1, 1990)
Printed on Recycled Paper
-------
SAMPLE C
PERMIT N0
COLLECTOR
V. SNOW (CIRCLE ONE)
AIR TEMP ('C). < Oytf^TtJl 11 ' 20'. > 21'
SAMPLE LOCATION SUPPLY WATER^gfASTEVVATffr RAW. TREATED
Other
PARAMETERS (CHECK APPROPRIATE)
B*cti
BOO
TSS
Turt) .
Organict
VOAi
NHJ
NOJ. 1
TKN
T P
O&G
CN
FLOW MEASUREMENT
None
Mtgmaltr
Vanluri
VNolCh
S»cl«ngul«r
METALjO
(•mpn
LAB CODE •
PROJECT fQ
STATION
SAMPLE
55SGJL
COLLECTION DATE
y MM D D Y YMM 0 D
START END
COLLECTION TIME
START END
SAMPLE TEMP CC)
SCntEABLE SOLIDS (m||)
FLOW METER
TOTALIZER
START
£NO
MULTIPLIER
l»m» Mil
(July 1, 1990)
-------
SAMPLE D
Veniuri
Panhali
V-NotCh
Rectangular
Other
COLLECTO
SNOW (CIRCLE ONE)
SOURCE
CITY
WEATHER
AIR TEMP CO <
SAMPLE LOCATION
Bacti
BOO
TSS
Turb
Organici
VOAi
FLOW MEASUREMENT
None
LAB CODE _W 55SCJ
PROJECTll I I I I | )
STATION f ^
SAMPLE
COLLECTION DATE
_ V_
YYMMOO
START
COLLECTION TIME
END
END
START
SAMPLE TEMP CC)
PM(SU)
TOTCIjJmg/l)
SETTLEABLE SOLIDS (m|/|)
.H
FLOW METER
TOTALIZER
START
END
MULTlPLIgH
to />#
(Sit.
(July 1, 1990)
-------
SAMPLE E
PERMIT NO
COLLECTOR _fi
WEATHER
AIR TEMP CO <
SAMPLE LOCATION SUPPLY WATER
(Circle One)
(CIRCLE ONE*
ASTEWATEIgRAW TREATED
Other
PARAMETERS (CHECK APPROPRIATE)
Bacti
BOO
TSS
Turb
Organici
VOAi
NH3
NOJ.
TKN
TP
OtC
CN
Other
FLOW MEASUREMENT
None
Megmeier
Venlun
Parthaii
V Notch
Rectangular
Other
SIZE
m
METALS
Toi
Cd
Cu
Cr(T)
'Or (+ 6)
Fa
Ng
Mn
Ni
Sn
tampie
LAB CODE
PROJECT fl
STATION I
SAMPLE
COLLECTION DATE
555 Cf
START
COLLECTION TIME
SAMPLE TEMP CC)
PH(SU)
1
FLOW METER
TOTALIZER
START
MULTIPLIER
ENO
ENO
tx
(July 1, 1990)
-------
EXAMPLE CHAIN OF CUSTODY RECORD FOR
SAMPLES A-E
(July 1, 1990)
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ATTACHMENT B
Daphnid (Ceriodaphnia dubia) Survival and Reproduction Test
and
Fathead Minnow (Pimephales promelas) Growth and Survival Test
(July 1, 1990) 11
-------
ATTACHMENT B
Chronic Toxicity Test Procedure and Protocol:
o Daphnid (Ceriodaphnia dubia) chronic (and modified acute)
static renewal survival and reproduction test.
o Fathead Minnow (Pimephales promelas) chronic (and modified
acute) static renewal larval growth and survival test.
I. GENERAL REQUIREMENTS
The permittee shall conduct acceptable toxicity tests in
accordance with the appropriate test protocols described below.
The permittee must collect discharge samples and perform the
toxicity tests that are required by Part I of the NPDES permit.
Chronic and acute toxicity data shall be reported as outlined in
Section IX. The chronic fathead minnow and daphnid tests can be
used to calculate the acute LC50 at the 48 hour exposure interval
where both an acute (LC50) and a chronic (C-NOEC) test is
specified in the permit.
II. TEST FREQUENCY AND SAMPLING REQUIREMENTS
See Part I of the NPDES permit for sampling location, sample
type, test frequency, test species, and test date(s)
requirements. Chain of Custody information should be provided
for each sample tested.
A chronic toxicity test sampling event is defined as three
discharge (composite or grab) samples collected over the seven-
day test period (see Section IV).
III. METHODS
'Methods to follow are those recommended by EPA in:
Weber, C.I., et al, 1989. Short Term Methods For Estimating The
Chronic Toxicity of Effluents and Receiving Water to Freshwater
Organisms. Second Edition. Office of Research and Development,
Cincinnati, OH, EPA/600/4-89/001.
Any exceptions are stated herein.
IV. SAMPLE COLLECTION
For each sampling event, three discharge samples shall be
collected over a 7-day exposure period. An initial sample (day
0) is used to start the test. The additional two samples are
collected for use at the start of day 3 and 5. Renewal of test
concentrations is conducted daily with the most recently
collected discharge sample. The initial (day 0) sample will be
analyzed chemically. Day 3 and 5 samples will be held until test
completion. If either the day 3 or 5 renewal sample is of
(July 1, 1990) 13
-------
sufficient potency to cause lethality to 50 percent or more test
organisms in any of the dilutions for either species, then a
chemical analysis shall be performed on the appropriate sample(s)
as well.
Aliquots shall be split from the samples, containerized and
preserved (as per 40 CFR Part 136) for chemical and physical
analyses. The remaining samples shall be dechlorinated (if
necessary) in the laboratory using sodium thiosulfate for
subsequent toxicity testing. Grab samples must be used for pH,
temperature, and total residual chlorine (as per 40 CFR Part
122.21).
The Methods for Aquatic Toxicity Identification Evaluations
(Phase I) EPA/600/3-88/034, Section 8.7, provides detailed
information regarding the use of sodium thiosulfate (i.e.
dechlorination).
All samples held overnight shall be refrigerated at 4°C.
V. DILUTION WATER
Dilution water used for chronic toxicity analysis shall be
collected at a point upstream of the discharge free from toxicity
or other sources of contamination. When using receiving water as
the dilution water an additional control (0% effluent), made up
from a standard dilution water of known quality, will also be
run.
If the receiving water diluent is found to be, or suspected to be
toxic or unreliable, an alternate surface water or standard
dilution water of known quality with a hardness, pH,
conductivity, alkalinity, and total suspended solids similar to
that of the receiving water may be substituted AFTER RECEIVING
WRITTEN APPROVAL FROM THE PERMIT ISSUING AGENCY(S). It may prove
beneficial to the permittee to have the proposed dilution water
•source screened for suitability prior to toxicity testing. For
further information see Section 7, page 24 of EPA/600/4-89/001.
(July 1, 1990) 14
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VI. REGION I RECOMMENDED EFFLUENT TOXICITV TEST CONDITIONS FOR
THE DAPHNID fCeriodaohnia dubia) SURVIVAL AND REPRODUCTION
TEST1
1. Test type:
2. Temperature (°C) :
3. Light quality:
4. Photoperiod:
5. Test chamber size:
6. Test solution volume:
7. Renewal of test solutions:
8. Age of test organisms:
9. Number of neonates per test
chamber:
10. Number of replicate test
chambers per treatment:
11. Number of neonates per test
concentration:
12. Feeding regime:
13. Aeration:
14. Dilution water:2
Static, renewal
25 ± 1°C
Ambient laboratory
illumination
16 hr. light, 8 hr. dark
30 ml
15 ml
Daily using most recently
collected sample.
Less than 24 hr.; and all
released within an 8 hr.
period of each other.
10
10
Feed 0.1 ml each of YTC and
algal suspension per exposure
chamber daily.
None
Receiving water, other surface
water, moderately hard
synthetic water (prepared
using either Millipore Milli-
QR or equivalent deionized
water and reagent grade
chemicals) or deionized water
combined with mineral water.
(July 1, 1990)
15
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15. Effluent concentrations:3
16. Dilution factor:
17. Test duration:
18. End points:
19. Test acceptability;
20. Sampling requirements:
21. Sample volume required:
5 concentrations and a
control. An additional
dilution at the permitted
effluent concentration
(% effluent) is required if
it is not included in the
dilution series.
0.5
Until 60% of control females
have three broods (may require
7 days).
Survival and reproduction
80% or greater survival and an
average of 15 or more
young/female in the control
solutions. At least 60% of
surviving females in controls
must have produced third
brood.
For on-site tests, samples are
collected daily and used
within 24 hr. of the time they
are removed from the sampling
device. For off-site tests, a
minimum of three samples are
collected (i.e. days 0, 3, 5)
and used for renewal (see Sec
IV). Off-site tests samples
must be first used within 48
hours of collection.
Minimum 2 liters/day.
Footnotes;
1. Adapted from EPA/600/4-89/001.
2. Standard dilution water must have hardness requirements to
generally reflect characteristics of the receiving water.
3. When receiving water is used for dilution an additional
control made up of standard dilution water (0% effluent) is
required.
(July 1, 1990)
16
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VII. REGION I RECOMMENDED EFFLUENT TEST CONDITIONS FOR THE
FATHEAD MINNOW fPimephales promelasl LARVAL SURVIVAL
AND GROWTH TEST
1. Test type:
2. Temperature (°C).:
3. Light quality:
4. Photoperiod:
5. Test chamber size:
6. Test solution volume:
7. Renewal of test
concentrations:
8. Age of test organisms:
9. No. larvae/test chamber
and control:
10. No. of replicate chambers/
concentration:
11. No. of larvae/concentration:
12. Feeding regime:
13. Cleaning:
14. Aeration:
Static, renewal
25 ± 1°C
Ambient laboratory
illumination
16 hr. light, 8 hr. dark
250 - 1000 ml
Minimum 200 ml/replicate
Daily using most recently
collected sample.
Newly hatched larvae less
than 24 hr. old
15 (minimum of 10)
4 (minimum of 3)
60 (minimum of 30)
•
Feed 0.1 ml newly hatched
brine shrimp nauplii twice
daily, 6 hrs. between feedings
(at the beginning of the work
day prior to renewal, and at
the end of the work day
following renewal).
Sufficient larvae are added to
provide an excess.
Siphon daily, immediately
before test solution renewal.
None, unless DO concentrations
falls below 40% saturation.
Rate should be less than 100
bubbles/min.
(July 1, 1990)
17
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15. Dilution water:2
16. Effluent concentrations:
17. Dilution factor:
18. Test duration:
19. End points:
20. Test acceptability:
21. Sampling requirements:
22. Sample volume required:
Receiving water, other surface
water, moderately hard
synthetic water (prepared
using either Millipore Milli-
QR or equivalent deionized and
reagent grade chemicals) or
deionized water combined with
mineral water.
5 and a control. An
additional dilution at
the permitted effluent
concentration (% effluent)
is required if it is not
included in the dilution
series.
0.5
7 days
Survival and growth (weight)
80% or greater survival in
controls: average dry weight
of controls equals or exceeds
0.25 mg
For on-site tests, samples are
collected daily and used
within 24 hours of the time
they are removed from the
sampling device. For off-
site tests, a minimum of three
samples are collected (i.e.
days 0, 3, 5) and used for
renewal (see Sec.IV). Off-
site tests samples must be
first used within 48 hours of
collection.
Minimum 2.5 liters/day.
Footnotes;
1. Adapted from EPA/600/4-89/001.
2. Standard dilution water must have hardness requirements to
generally reflect characteristics of the receiving water.
3. when receiving water is used for dilution an additional
control made up of standard dilution water (0% effluent) is
required.
(July 1, 1990)
18
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VIII. CHEMICAL ANALYSIS
The following chemical analyses shall be performed for each
sampling event.
Minimum
Detection
Parameter Effluent Diluent Limit (rag/1)
Hardness*1
Alkalinity
PH
Specific Conductance
Total Solids and Suspended Solids
Ammonia
Total Organic Carbon
Total Residual Chlorine (TRC)
•2
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
0
2
-
-
-
0
0
0
-5 .
.0
-
-
-
.1
.5
.02
Total Metals
Cd
Cr, Ni
Pb, Cu
Zn, Al
Mg, Ca
x
x
X
X
X
X
X
X
0.005
0.05
0.005
0.02
0.05
In addition, the following chemical analyses shall be performed
as part of each daily renewal procedure on each dilution and the
controls.
Parameter
Dissolved Oxygen
Temperature
PH
Specific Conductance
Alkalinity
Hardness
Superscripts:
Beginning of 24-hr
Exposure Period
End of 24-hr
Exposure Period
x
x
X
X
•1
Method 314 A (hardness by calculation) from APHA (1985)
Standard Methods for the Examination of Water and
Wastewater. 16th Edition.
Total
Residual Chlorine
either of the following methods from the 16th
(1985) Standard Methods for the
for these
Methods:
Edition of the APHA
Examination of Water and Wastewater must be used
analyses:
Method 408-C (Amperometric Titration Method)-the
preferred method;
Method 408-D (Ferrous Titrimetric Method).
These are performed on the'100% e'ffluent and control samples
only.
(July 1, 1990)
19
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IX. TOXICITY TEST REPORT ELEMENTS
A report of results will include the following:
Description of sample collection procedures, site
description;
Names of individuals collecting and transporting samples,
tines and dates of sample collection and analysis; and
General description of tests: age of test organisms,
origin, dates and results of standard toxicant tests,' light
and temperature regime; other information on test conditions
if different than procedures recommended.
Toxicity test data shall include the following:
Chronic
Daily survival of test organisms in the controls and all
replicates in each dilution. Survival data should be
analyzed by Fisher's Exact Test prior to analysis of
reproduction data.
Young per female for all replicates in each dilution for
Ceriodaphnia and weight for minnow larvae.
Chronic test data shall undergo hypothesis testing to
determine if the distribution of results is normal using the
Shapiro-Wilks test. The variance must also be tested for
homogeneity using Bartlett's Test. Then the endpoint
estimates, NOEC and LOEC must be determined using Dunnett's
Procedure, Bonferroni's T-Test, Steel's Many-One Rank Test,
or Wilcoxon Rank Sum Test. The choice of test depends on
the number of replicates and whether the variance is
homogeneous or not. See EPA/600/4-87/028 for details. (Any
printouts and graphical displays must be submitted, along
with the name of the program, the date, and the author(s).
When data is analyzed by hand, worksheets should be
included.)
C-NOEC: Chronic-No Observed Effect Concentration.
LOEC: Lowest Observed Effect Concentration.
MATC: Maximum Allowable Toxicant Concentration.
Acute (These data points are to be obtained 48-hrs into the
chronic test.)
Survival for each concentration and replication at time 24,
and 48 hours; and
LC50 and 95% confidence limits using one of the following
methods in order of preference, Probit, Trimmed Spearman
Karber, Moving Average Angle, or Graphical method; printout
or copy of these calculations.
The Probit, Trimmed Spearman Karber, and Moving Average
Angle methods of analyses can only be used when mortality of
some of the test organisms are observed in at least two of
the (% effluent) concentrations tested (i.e. partial
mortality). If a test results in a 100% survival and
100% mortality in adjacent treatments ("all or nothing"
effect), a LC50 may be estimated using the graphical method.
(July 1, 1990) 20
-------
Chronic and Acute
All chemical/physical data generated. (Include detection
limits.)
Raw data and bench sheets. (See sample data sheets, pp. 22-
29.)
Provide a description of dechlorination procedures (as
applicable).
Any other observations or test conditions affecting test
outcome.
X. REPORTING
Signed copies of the toxicity testing reports shall be submitted
as required by Part I of the permit.
(July 1, 1990) 21
-------
Data form for the Cen'odaphm'a survival and reproduction test.
Daily record.
(The chart on tne right was reduced to save space)
Discharger:
Location:
Date Sample Collected:
Analyst:
Test Dates:
Template No.:
Dilution Water:
/ * Test organism alive
x - Test organism dead
0 = Number of live young
(-0) = Number of dead young
M = Lost or missing
y * Male
Test Chambers (glass/plastic)
Food:
Test Temp:
Test Organisms (age):
Comments:
I
1
1
'
1
t
1
•
1
1
1
1
1
1
i !
•
0
'
1
I
1 ,
i ]
m
j
l
< i
•
•
17
•
37
»
M
M
• 7
5'
II
!
1
j
1
* j
(July 1, 1990)
22
-------
Data form for the Ceriodaphm'a survival and reproduction test.
Summary of data
Discharger:
Location:
Date Sample Collected:
Analyst:
Test Start-Date/Time:
Test Stop -Date/Time:"
Cone
Day
1
2
3
4
5
6
7
8
Total
Replicate
1
2
J
4
5
6
/
B
9
10
No. Of
Young
No. of
Adul ts
Young per
Adult
.
Cone.
Day
1
2
3
4
5
6
7
8
Total
1
2
J
1
4
leph
i
catt
b
i
)
tt
9
IU
NO. Of
Young
NO. of
Adults
Young per
Adult
Cone.
Day
1
2
3
4
5
5
7
8
Total
1
2
J
[
4
tepli
I
catc
t
i
J
0
9
10
NO. of
Young
No. of
Adults
Young per
Adult
(July 1, 1990)
23
-------
Data form for the Cen'odaphm'a survival and reproduction test.
Summary of data (Continued)
Cone.
Replicate
Day
1
2
3
4
5
6
7
8
Total
1
I
3
4
5
6
;
d
9
10
No. of
Young
No. of
Adults
Young per
Adult
Cone.
Replicate
Day
l
2
3
4
b
6
7
0
Total
1
2
J
4
b
b
/
8
9
10
No. Of
Young
No. of
'Adults
Young per
Adult
Cone.
Reolicate
Day
1
2
3
4
5
6
7
8
Total
1
2
J
4
b
b
;
B
9
10
No. Of
Young
NO. of
'Adults
Young per
Adult
(July 1, 1990)
24
-------
Discharger:
Location:
Data fonn for the fathead minnow larval survival
and growth test. Routine chemical and physical
determinations.
Test Dates:_
Analyst:
Control :
etnp.
0.0. Initial
Final
pH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
^
4
6
7
Remarks
Cone:
Temp.
D.D. initial
Final
pH initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
Da.
1
Z
1
4
j
5
6
7
Remarks
Daj
Cone:
Temp
D.O. Initial
Final
oH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
1
Z
3
4
1
5
•
6
7
Remarks
(July 1, 1990)
25
-------
xRrn.
Data form for the fathead minnow larval survival and growth
test. Routine chemical and physical determinations.
(Continued).
Discharger:
Location:
Test Dates:
Analyst:
Cone:
Temp.
D.D. Initial
Final
pH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
Da
3
4
f
5
I'
... 1
Remarks
Cone:
Temp.
D.O. Initial
Final
pH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
Daj
1
Z
3
4
f
5
0
7
Remarks
Cone:
Temp.
D.O. Initial
Final
DH Initial
Final
Alkalinity
Hardness
Conductivity
Chlorine
Daj
1
7»
1
T^
•)
6
Remarks
(July 1, 1990)
26
-------
Discharger:
Location:
Survival data for fathead minnow larval survival and growth test.
Test Dates:
Analyst:
Cone: Rep.
No.
Control
Cone:
Cone:
Cone:
Cone:
Cone:
No. Survivors
Da>
1
2
3
4
5
6
7
Remarks
Comments:
(July 1, 1990)
27
-------
Weight data for fathead minnow larval survival and growth test
c
I-*
K:
\0
\o
o
Discharge:
Location: ~
Analyst:
Test Uate(s):
Weighing Date:'
Drying Temperature (°C)
Drying Time (h):
Cone :
Control
Cone :
Cone :
Cone :
Lone :
Cone :
Kep.
No.
A
Wgt. of
boat
<«g)
B
Dry wgt:
foil and
larvae
(ing)
B-A
Total dry
wgt of
1 a rvae
(nig)
C
No. of
larvae
(B-A)/C
Mean dry wgt
of larvae
(mg)
Remarks
N)
CD
Adapted from Hughes, et al., 1967.
-------
Discharger:
Location:
Summary data for fathead minnow larval survival
and growth test.1
Test Dates:
Analyst:
Treatment
No. live
larvae
Survival
(%)
Mean dry wot
of larvae (mg)
* SO
Temperature
range (°C)
Dissolved
oxygen range
(mg/L)
Hardness
Conductivity
Control
Comments:
(July l, 1990)
29
-------
ATTACHMENT C
Daphnid (Ceriodaphnia dubia and Daphnia pulex)
and
Fathead Minnow (Pimephales promelas) 48 Hour Acute Tests
(July 1, 1990) 31
-------
ATTACHMENT C
Acute Toxicity Test Procedure and Protocol:
0 Daphnids (Ceriodaphnia dubia and Daphnia pulex) definitive
48 hour acute test.
0 Fathead Minnow (Pimephales promelas) definitive 48 hour
acute test.
•I. GENERAL REQUIREMENTS
The permittee shall conduct acceptable toxicity tests in
accordance with the appropriate test protocols described below.
The permittee must collect discharge samples and perform the
toxicity tests that are required by Part I of the NPDES permit.
Acute toxicity test data shall be reported as outlined in Section
IX.
II. TEST FREQUENCY AND SAMPLING REQUIREMENTS
See Part I of the NPDES permit for sampling location, sample
type, test frequency, test species, and test date(s)
requirements. Chain of Custody information should be provided
for each sample tested.
An acute toxicity test sampling event is defined as a single
discharge (composite or grab) sample.
III. METHODS
Methods should follow those recommended by EPA in:
Peltier, W., and Weber, C.I., 1985. Methods for Measuring the
Acute Toxicitv of Effluents to Freshwater and Marine Organises.
Third Edition. Office of Research and Development, Cincinnati,
OH. EPA/600/4-85/013.
Any exceptions are stated herein.
IV. SAMPLE COLLECTION
A discharge sample shall be collected. Aliquots shall be split
from the sample, containerized and preserved (as per 40 CFR
Part 136) for chemical and physical analyses required. The
remaining sample shall be dechlorinated*(if necessary) in the
laboratory using sodium thiosulfate for subsequent toxicity
testing. Grab samples must be used for pH, temperature, and
total residual chlorine (as per 40 CFR Part 122.21).
The Methods for Aquatic Toxicitv Identification Evaluations
rPhase I) EPA/600/3-88/034, Section 8.7, provides detailed
information regarding the use of sodium thiosulfate (i.e.
dechlorination).
All samples held overnight shall be refrigerated at 4°C.
(July 1, 1990) 33
-------
V. DILUTION WATER
Dilution water used for acute toxicity analysis shall be
collected at a point upstream of the discharge free from toxicity
or other sources of contamination. When using receiving water as
the dilution water an additional control (0% effluent), made up
from a standard dilution water of known quality, will also be
run.
If the receiving water diluent is found to be, or suspected to be
toxic or unreliable, an alternate standard dilution water of
known quality with a hardness, pH, conductivity, alkalinity, and
total suspended solids similar to that of the receiving water may
be substituted AFTER RECEIVING WRITTEN APPROVAL FROM THE PERMIT
ISSUING AGENCY(S). It may prove beneficial to the permittee to
have the proposed dilution water source screened for suitability
prior to toxicity testing. For further information see
Section 6, page 22 of EPA/600/4-85/013.
(July 1, 1990) 34
-------
VI. REGION I RECOMMENDED EFFLUENT TOXICITY TEST CONDITIONS FOR
THE DAPHNIDS fCeriodaphnia dubia and Daphnia pulex) 48 HOUR
ACUTE TESTS
1. Test type
2. Temperature (°C)
3. Light quality
4. Photoperiod
5. Test chamber size
6. Test solution volume
7. Age of test organisms
8. No. daphnids per test chamber
9. No. of replicate test chambers
per treatment
10. Total no. daphnids per test
concentration
11. Feeding regime
12. Aeration
13. Dilution water2
14. Dilution factor
15. Number of dilutions3
16. Effect measured
Static, non-renewal
25 ± 1°C
Ambient laboratory
illumination
16 hour light, 8 hour dark
Minimum 30 ml
Minimum 25 ml
1-24 hours (neonates)
5
4
20
None
None
Receiving water, other surface
water, moderately hard
synthetic water (prepared
using either Millipore Milli-
QR or equivalent deionized
water and reagent grade
chemicals) or deionized water
combined with mineral water.
0.5
5 plus a control. An
additional dilution at the
permitted effluent
concentration (% effluent)
is required if it is not
included in the dilution
series.
Mortality - no movement of
body or appendages on gentle
prodding
(July 1, 1990)
35
-------
17. Test acceptability
18. Sampling requirements
19. Sample volume required
90% or greater survival of
test organisms in control
solution
For on-site tests, samples
must be used within 24 hours
of the time that they are
removed from the sampling
device. For off-site tests,
samples must first be used
within 48 hours of collection,
Minimum 2 liters
Footnotes:
1. Adapted from EPA/600/4-85/013.
2. Standard prepared dilution water must have hardness
requirements to generally reflect the characteristics of the
receiving water.
3. When receiving water is used for dilution an additional
control made up of standard dilution water (0% effluent) is
required.
(July 1, 1990)
36
-------
VII. REGION I RECOMMENDED TEST CONDITIONS FOR THE FATHEAD MINNOW
(Pimephales promelas) 48 HOUR ACUTE TEST1
1. Test Type:
2. Temperature (°C) :
3. Light quality:
4. Photoperiod:
5. Size of test vessels:
6. Volume of test solution:
7. Age of fish:
8. No. of fish per chamber:
9. No. of replicate test vessels
per treatment:
10. Total no. organisms per
concentration:
11. Feeding regime:
12. Aeration:
13. Dilution Water:2
14. Dilution factor:
Static, non-renewal
25 ± i°c
Ambient laboratory
illumination
16 hr light, 8 hr dark
250-1000 ml
Minimum 200ml/replicate
1-14 days
10 (not to exceed loading
limits)
20
None
None, unless DO concentration
falls below 40% of saturation,
at which time gentle single-
bubble aeration should be
started at a rate of less than
100 bubbles/min. (Routine DO
check recommended.)
Receiving water, other surface
water, moderately hard
synthetic water (prepared
using either Millipore Milli-
QR or equivalent deionized and
reagent grade chemicals) or
deionized water combined with
mineral water.
0.5
(July 1, 1990)
37
-------
15. Number of dilutions:3
16. Effect measured:
17. Test acceptability;
18. Sampling requirements:
19. Sample volume required:
5 plus a control. An
additional dilution at the
permitted effluent
concentration (% effluent)
is required if it is not
included in the dilution
series.
Mortality-no movement on
gentle prodding
90% or greater survival of
test organisms in control
solution
For on-site tests, samples
must be used within 24
hours of the time that
they are removed from
the sampling device. For
off-site tests, samples
must be first used within 48
hours of collection.
Minimum 4 liters
Footnotes;
1. Adapted from EPA/600/4-85/013.
2. Standard dilution water must have hardness requirements to
generally reflect characteristics of the receiving water.
3. When receiving water is used for dilution, an additional
control made up of standard dilution water (0% effluent) is
required.
(July 1, 1990)
38
-------
VIII. CHEMICAL ANALYSIS
The following chemical analyses shall be performed for each
sampling event.
Minimum
Detection
Parameter Effluent Diluent Limit (nq/L)
Hardness x
Alkalinity x
pH x
Specific Conductance x
Total Solids and Suspended Solids x
Ammonia x
Total Organic Carbon f x
Total Residual Chlorine (TRC) x
Total Metals
Cd x
Cr, Ni x
Pb, Cu x
Zn, Al x
Mg, Ca x
Superscripts:
x
x
X
X
X
X
X
X
X
X
X
0.5
2.0
0.1
0.5
0.02
0.005
0.05
0.005
0.02
0.05
1 Method 314A (hardness by calculation) from APHA (1985)
Standard Methods for the Examination of Water and
wastewater. 16th Edition.
*2 Total Residual Chlorine
Methods: either of the following methods the 16th edition
of the APHA (1985) Standard Methods for the Examination of
Water and Wastewater must be used for these analyses.
Method 408-C (Amperometric Titration Method)-the
preferred method;
Method 408-D (Ferrous Titrimetric Method).
IX. TOXICITY TEST REPORT
The following must be reported:
Description of sample collection procedures, site
description;
Names of individuals collecting and transporting samples,
times and dates of sample collection and analysis; and
General description of tests: age of test organisms,
origin, dates and results of standard toxicant tests; light
and temperature regime; other information on test conditions
if different than procedures recommended.
(July 1, -1990)
39
-------
Toxicity test data shall include the following:
Survival for each concentration and replication at time 24,
and 48 hours.
LC50 and 95% confidence limits shall be calculated using one
of the following methods in order of preference Probit,
Trimmed Spearman Karber,'Moving Average Angle, or the
graphical method. All printouts (along with the name of the
program, the date, and the author(s)) and graphical displays
must be submitted. When data is analyzed by hand,
worksheets should be submitted.
The Probit, Trimmed Spearman Karber, and Moving Average
Angle methods of analyses can only be used when mortality of
some of the test organisms are observed in at least two of
the (% effluent) concentrations tested (i.e. partial
mortality). If a test results in a 100% survival and
100% mortality in adjacent treatments ("all or nothing"
effect), a LC50 may be estimated using the graphical method.
All chemical/physical data generated (include detection
limits).
Raw data and bench sheets. (See sample data sheets, pp. 41-
42.)
Describe method of dechlorination where applicable.
Any observations and test conditions which affected the
outcome of testing.
X. REPORTING
Signed copies of the toxicity testing reports shall be submitted
as required by of Part I of the NPDES permit.
(July 1, 1990) 40
-------
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(-1
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IO
1. EXPOSURE CHftHBER
Total vessel capacity:
lest solution voluae:
Mater depth: constant -
cjcltc -
3. ACRA1IOM
None:
Slow:
Hoderate:
CM
to
CM.
Beginning
b. Condition of surviving orgontsos *t end of test:
2. FEEDING SCHEDUIE
Not fed:
Fed daily:
Fed irregularly (describe):
4. SCREENED ANIHM. ENCLOSURES
Not used:
Used:
(CM) Dtaveter
6. CoMienls:
Check list OD back of effluent toxtcity data sheet.
-------
ATTACHMENT D
Sea Urchin (Arbacia punctulata) Fertilization Test
and
Algal (Champia parvula) Reproduction Test
(July 1, 1990) 43
-------
ATTACHMENT D
chronic Toxicity Test procedure and Protocol:
0 Sea Urchin (Arbacia punctulata) 1 hour fertilization test.
0 Algal (Champia parvula) 2 day exposure, 5 to 7 day recovery,
reproduction test.
I. INTRODUCTION
The permittee shall conduct acceptable toxicity tests in
accordance with the appropriate test protocols described below.
The permittee must collect discharge samples and perform toxicity
tests that are required by Part I of the NPDES permit. Chronic
toxicity data shall be reported as outlined in Section IX.
II. TEST FREQUENCY AND SAMPLING REQUIREMENTS
See Part I of the NPDES permit for sampling location, sample
type, test species, and test date(s) requirements. Chain of
Custody information should be provided for each sample tested.
A sampling event is defined as a single discharge (composite or
grab) sample.
III. METHODS
Methods to follow are those recommended by EPA in:
Weber, C.I., et al, 1988. Short Term Methods for Estimating the
Chronic Toxicitv of Effluents and Receiving Waters To Marine and
Estuarine Organisms. Office of Research and Development, Cincin-
nati, OH. EPA/600/4-87/028.
Any exceptions are stated herein.
IV. SAMPLE COLLECTION
A discharge sample shall be collected. Aliquots shall be split
from the sample, containerized and preserved (as per 40 CFR
Part 136) for the chemical and physical analyses. The remaining
sample shall be dechlorinated (if necessary) in the laboratory
using sodium thiosulfate for subsequent toxicity testing. Grab
samples must be used for pH, temperature, and total residual
oxidants (as per 40 CFR Part 122.21).
The Methods for Aquatic Toxicitv Identification Evaluations
fPhase I) EPA/600/3-88/034, Section 8.7 provides detailed
information regarding the use of sodium thiosulfate (i.e.
dechlorination).
All samples held overnight shall be refrigerated at 4°C.
{July 1, 1990) 45
-------
V. DILUTION WATER
Dilution water used for toxicity analysis shall be collected at a
point away from the discharge which is free from toxicity or
other sources of contamination. When using receiving water as
the dilution water an additional control (0% effluent), made up
from a standard dilution water of known quality, will also be
run.
If the receiving water diluent is found to be, or suspected to be
toxic or unreliable, an alternate surface water or standard
dilution water of known quality with a conductivity, salinity,
total suspended solids, and pH similar to that of the receiving
water may be substituted AFTER RECEIVING WRITTEN APPROVAL FROM
THE PERMIT ISSUING AGENCY(S). It may prove beneficial to the
permittee to have the proposed dilution water source screened for
suitability prior to toxicity testing. For further information,
see Section 7, page 19 of EPA/600/4-87/028.
Dilution water used for marine chronic toxicity shall be of
sufficient quality to meet minimum acceptability of test results.
(See Sections VI and VII.)
For Arbacia punctulata. an uncontaminated source of natural
seawater, deionized water mixed with hypersaline brine, or
artificial sea salts with a salinity of 30 o/oo + 2 o/oo must be
used.
For Champia parvula. an uncontaminated source of natural seawater
(30 o/oo S) or a combination of 50 percent 30 o/oo salinity
natural seawater and 50 percent 30 o/oo salinity artificial
seawater must be used.
(July 1, 1990) 46
-------
VI. REGION I RECOMMENDED TEST CONDITIONS FOR THE SEA URCHIN
fArbacia punctulata) FERTILIZATION TEST1
1.
2.
3.
4.
Test type
Salinity
Temperature
Light quality
Static, non-renewal
30 o/oo ± 2 o/oo
20 ± 1°C
Ambient laboratory light
7.
8.
9.
10.
11,
Light intensity
Test vessel size
Test solution volume
Number of sea urchins
Number of egg and sperm cells
per chamber
Number of replicate chambers
per treatment
Dilution water
13
14
Dilution factor
Test duration
Effects measured
15. Number of treatments per
test2
during test preparation
10-20 uE/m2/s, or 50-100 ft-c
(Ambient Laboratory Levels)
Disposable (glass) liquid
scintillation vials (20 ml
capacity), not pre-cleaned
5 ml
Pooled sperm from four males
and pooled eggs from four
females are used per test
About 2000 eggs and 5,000,000
sperm cells per vial
4 (minimum of 3)
Uncontaminated source of
natural seawater; deionized
water mixed with hypersaline
brine or artificial sea salts
0.5
1 hour and 20 minutes
Fertilization of sea urchin
eggs
5 and a control. An
additional dilution at the
permitted effluent
concentration (% effluent)
is required.
(July 1, 1990)
47
-------
16. Acceptability of test
results
17. Sampling requirements
18. Sample volume required
Recommended sperm:egg ratio
must result in fertilization
of minimum of 70% of the eggs
in the control chambers.
For on-site tests, samples are
to be used within 24 hours of
the time that they are removed
from the sampling device. For
off-site tests, samples must
be first used within 48 hours
of collection.
Minimum 2 liters.
Footnotes;
1. Adapted from EPA/600/4-87/028.
2. When receiving water is used for dilution an additional
control made up of standard dilution water (0% effluent) is
required.
(July 1, 1990)
48
-------
VII. REGION I RECOMMENDED TEST CONDITIONS FOR THE ALGAL (Chanpia
oarvula) SEXUAL REPRODUCTION TEST1
1. Test Type
2. Salinity
3. Temperature
4. Photoperiod
5. Light intensity
6. Light source
7. Test chamber
8. Test solution volume
9. Dilution water
10. Dilution factor
11. Number of dilutions2
12. Number of replicate chambers
per treatment
13. Number of organisms per
test chamber
14. Test duration
15. Effects measured
Static, non-renewal
30 o/oo + 2 o/oo
22 - 24°C
16 hr. light, 8 hr. dark
100 uE/m2/s (500 ft-c)
Cool-white fluorescent lights
200 ml polystyrene cups, or
250 ml Erlenmeyer flasks
100 ml
30 o/oo salinity natural
seawater, or a combination of
50% 30 o/oo salinity natural
seawater and 50% 30 o/oo
salinity artificial seawater.
0.5
5 and a control. An
additional dilution at the
permitted effluent
concentration (% effluent)
is required.
4 (minimum of 3)
5 female branch tips and
1 male plant
2-day exposure to effluent,
followed by 5- to 7-day
recovery period in control
medium for cystocarp develop-
ment.
Reduction in cystocarp produc-
tion compared to controls
(July 1, 1990)
49
-------
16. Acceptability of test
17. Sampling requirements
18. Sample volume
Control mortality must not
exceed 20%.
Plant fragmentation in the
controls or lower exposure
concentrations should be
minimal.
Control plants must average 10
or more cystocarps.
For on-site tests, samples
must be used within 24 hours
of the time they are removed
from the sampling device.
Off-site test samples must
be used within 48 hours of
collection.
Minimum of 2 liters.
Footnotes;
1. Adapted from EPA/600/4-87/028.
2. When receiving water is used for dilution an additional
control made up of standard dilution water (0% effluent) is
required.
(July 1, 1990}
50
-------
VIII. CHEMICAL ANALYSIS
The following chemical analyses shall be performed for each
sampling event.
Minimum
Detection
Parameter Effluent Diluent Limit(mq/L)
x
x
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
PPT(o/oo)
0.02
0. 1
0.5
0.01
0.05
0.01
0.02
PH
Specific Conductance
Salinity
Total Residual Oxidants1
Total Solids and Suspended Solids
Ammonia
Total Organic Carbon
Total Metals
Cd
Cr, Ni
Pb, Zn, Cu
Al
Superscripts:
1 Total Residual Oxidants
Methods: either of the following methods from the 16th
Edition of the APHA (1985) Standard Methods for the
Examination of Water and Wastewater must be used for these
analyses:
Method 408-C (Amperometric Titration Method)-the
preferred method;
Method 408-D (Ferrous Titrimetric Method).
IX. TOXICITY TEST REPORT ELEMENTS
A report of results will include the following:
Description of sample collection procedures, site
description;
Names of individuals collecting and transporting samples,
times and dates of sample collection and analysis; and
General description of tests: age of test organisms,
origin, dates and results of standard toxicant tests; light
and temperature regime; other information on test conditions
if different than procedures recommended.
(July 1, 1990)
51
-------
Toxicity test data shall include the following:
Daily survival of test organisms in the controls and all
replicates in each dilution if applicable.
Chronic test data shall undergo hypothesis testing to
determine if the distribution of results is normal using the
Shapiro-wilks test. The variance must also be tested for
homogeneity using Bartlett's Test. Then the endpoint
estimates, NOEC and LOEC must be determined using Dunnett's
Procedure, Bonferroni's T-Test, Steel's Many-One Rank Test,
or Wilcoxon Rank Sum Test. The choice of test depends on
the number of replicates and whether the variance is
homogeneous or not. See EPA/600/4-87/028 for details.
(All printouts and graphical displays must be submitted,
along with the name of the program, the date, and the
author(s). When data is analyzed by hand, the worksheets
should be submitted.)
C-NOEC: Chronic-No Observed Effect Concentration.
LOEC: Lowest Observed Effect Concentration.
MATC: Maximum Allowable Toxicant Concentration.
All chemical/physical data generated (include detection
limits).
Raw data and bench sheets. (See sample data sheets, pp. 53-
57.)
Any test conditions and observations affecting test outcome.
X. REPORTING
Signed copies of the toxicity testing reports shall be submitted
as required by Part I of the permit.
(July 1, 1990) 52
-------
Data sheet for Champ fa parvula sexual reproduction test.
Receiving water sunwary sheet.
SITE
COLLECTION DATE_
TEST DATE
LOCATION
INITIAL
SALINITY
FINAL
SALINITY
SOURCE OF SALTS FOR
SALINITY ADJUSTMENT'
1 • natural Mawttar, QP2 Done. QP2 talts, tic
(include ton* indication of amount)
COMMENTS
(July 1, 1990)
S3
-------
Data sheet for Cham 1 a parvula sexual reproduction test.
Cystocarp data shee
p 1
L
COLLECTION DATE
EXPOSURE BEGAN (date)
RECOVERY BEGAN «Jate)_
COUNTED (date)
EFFLUENT OR TOXICANT
TREATMENTS (% EFFLUENT, ^G/L, or REC. WATER SITES)
REPLICATES
CONTROL
A 1
2
3
4
MEAN
,
B 1
2
3
4
MEAN
C 1
2
3
' 4
MEAN
OVERALL
MEAN
Temperature
1
. i
Salinity
.ight
Source ot Dilution Water
. /* ^ 1 1 ft OT
(July l, 1990)
54
-------
Data sheet for (1) fertilization test using Arbacia punctulata.
TEST DATE:
SAMPLE:
COMPLEX EFFLUENT SAMPLE:
COLLECTION DATE:
SALINITY/ADJUSTMENT: __
PH/ADJUSTMENT REQUIRED:
PHYSICAL CHARACTERISTICS:
STORAGE:
COMMENTS:
SINGLE COMPOUND:
SOLVENT (CONC):
TEST CONCENTRATIONS:
DILUTION WATER:
CONTROL WATER:
TEST TEMPERATURE:
TEST SALINITY: _
COMMENTS:
(July 1, 1990) 55
-------
Data sheet (2) for fertilization test using Arbacia punctulata
TEST DATE: '
SAMPLE:
SPERM DILUTIONS:
HEMACYTOMETER COUNT, E: x 104 » SPM SOLUTION E «
SPERM CONCENTRATIONS: SOLUTION E x 40 « SOLUTION A » SPM
SOLUTION E x 20 » SOLUTION B • SPM
SOLUTION E x 5 - SOLUTION C • SPM
SOLUTION SELECTED FOR TEST ( - 5 x 10? SPM):
DILUTION: SPM/(5 x 10?) • DF
[(OF) x 5) - 5 • * SW, ML
FINAL SPERM COUNTS «
EGG DILUTIONS:
INITIAL EGG COUNT •
ORIGINAL EGG STOCK CONCENTRATION • 10X (INITIAL
EGG COUNT)
VOLUME OF SW TO ADD TO DILUTE EGG STOCK TO 2000/nt:
(EGG COUNT) - ZOO *
CONTROL WATER TO ADD EGG STODK. ML •
FINAL EGG COUNT •
TEST TIMES:
SPERM COLLECTED:
EGGS COLLECTED: .
SPERM ADDED:
EGGS ADDED:
FIXATIVE ADDED:
SAMPLES READ:
(July 1, 1990)
56
-------
Data sheet (3) for fertilization test using Arbacia punctulata.
DATE TESTED:.
SAMPLE:
TOTAL AND UNFERTILIZED EGG COUNT AT END OF TEST:
EFFLUENT
CONC (»>
REPLICATE VIAL
1
TOTAL-UNFERT
TOTAL-UNFERT
TOTAL-UNFERT
TOTAL-UNFERT
STATISTICAL ANALYSIS:
ANALYSIS OF VARIANCE:
CONTROL:
DIFFERENT FROM CONTROL (P):
COMMENTS:
(July 1, 1990)
57
-------
ATTACHMENT E
Mysid shrimp (Mvsidopsis bahia)
and
Inland Silverside (Menidia bervllina) Acute Tests
(July 1, 1990) 59
-------
ATTACHMENT E
Acute Toxicity Test Procedure and Protocol:
o Mysid Shrimp (Mvsidopsis bahia) definitive 48 hour test.
o Inland Silverside (Menidia bervllina) definitive 48 hour
test.
I. GENERAL REQUIREMENTS
The permittee shall conduct acceptable toxicity tests in
accordance with the appropriate test protocols described below.
The permittee shall collect discharge samples and perform the
toxicity tests that are required by Part I of the NPDES permit.
Acute toxicity data shall be reported as outlined in Section IX.
II. TEST FREQUENCY AND SAMPLING REQUIREMENTS
See Part I of the NPDES permit for sampling location, sample
type, test frequency, test species, and test date(s)
requirements. Chain of Custody information should be provided
for each sample tested.
A sampling event is defined as a single discharge (composite or
grab) sample.
III. METHODS
Methods to follow are those recommended by EPA in:
Peltier, W., and Weber, C.I., 1985. Methods for Measuring the
Acute Toxicity of Effluents to Freshwater and Marine Organisms.
Third Edition. Office of Research and Development, Cincinnati,
OH. EPA/600/4-85/013.
Any exceptions are stated herein.
IV. SAMPLE COLLECTION
A discharge sample shall be collected. Aliquots shall be split
from the sample, containerized and preserved (as per 40 CFR
Part 136) for the chemical and physical analyses. The remaining
sample shall be dechlorinated (if necessary) in the laboratory
using sodium thiosulfate for subsequent toxicity testing. Grab
samples must be used for pH, temperature, and total residual
oxidants (as per 40 CFR Part 122.21).
The Methods for Aquatic Toxicitv Identification Evaluations
(Phase I) EPA/600/3-88/034, Section 8.7, provides detailed
information regarding the use of sodium thiosulfate
dechlorination).
All samples held overnight shall be refrigerated at 4°C.
(July 1, 1990) 61
-------
V. DILUTION WATER
Dilution water used for acute toxicity analysis shall be
collected at a point away from the discharge which is free from
toxicity or other sources of contamination. When using receiving
water as the dilution water an additional control (0% effluent),
made up from a standard dilution water of known quality, will
also be run.
If the receiving water diluent is found to be, or suspected to be
toxic or unreliable, an alternate surface water or standard
dilution water of known quality with a conductivity, salinity,
total suspended solids, and pH similar to that of the receiving
water may be substituted AFTER RECEIVING WRITTEN APPROVAL FROM
THE PERMIT ISSUING AGENCY(8). It may prove beneficial to the
permittee to have the proposed dilution water source screened for
suitability prior to toxicity testing. For further information,
see Section 6, page 22 of EPA/600/4-85/013.
(July 1, 1990) 62
-------
VI. REGION I RECOMMENDED EFFLUENT TOXICITY TEST CONDITIONS FOR
THE MYSID (Mvsidopsis bahia) 48 HOUR TEST
1. Test type
2. Salinity
3. Temperature (°C)
4. Light quality
5. Photoperiod
*6. Test chamber size
7. Test solution volume
8. Age of test organisms
9. No. Mysids per test chamber
10. No. of replicate test chambers
per treatment
11. Total no. Mysids per test
concentration
12. Feeding regime
Static, non-renewal
25 ppt + 10 percent for all
dilutions
25°C
Ambient laboratory
illumination
16 hour light, 8 hour dark
250 ml
200 ml
1-5 days
10
13
14
Aeration
Dilution water
15,
16,
Dilution factor
Number of dilutions3
17. Effect measured
20
Light feeding (2 drops con-
centrated brine shrimp
nauplii, approx 100 nauplii/
mysid) twice daily
None
Natural seawater, synthetic
salt water, or deionized
water mixed with hypersaline
brine.
0.5
5 plus a control. An
additional dilution at the
permitted effluent
concentration (% effluent)
is required if it is not
included in the dilution
series.
Mortality - no movement of
body or appendages on gentle
prodding
(July 1, 1990)
63
-------
18. Test acceptability
19. Sampling requirements
20. Sample volume required
90% or greater survival of
test organisms in control
solution
For on-site tests, samples are
used within 24 hours of the
time that they are removed
from the sampling device. For
off-site tests, samples must
be first used within 48 hours
of collection.
Minimum 4 liters
Footnotes;
1. Adapted from EPA/600/4-85/013.
2. If dissolved oxygen falls below 40% saturation, aerate at
rate of less than 100 bubbles/min. Routine DO checks are
recommended.
3. When receiving water is used for dilution an additional
control made up of standard dilution water (0% effluent) is
required.
(July l, 1990)
64
-------
VII. REGION I RECOMMENDED TOXICITY TEST CONDITIONS FOR THE
INLAND SILVERSIDE (Menidia bervllina) 48 HOUR TEST1
1. Test Type
2. salinity
3. Temperature
4. Light Quality
5. Photoperiod
6. Size of test vessel
7. Volume of test solution
8. Age of fish
9. No. fish per chamber
10. No. of replicate test vessels
per treatment
11. Total no. organisms per con-
centration
12. Aeration2
13. Dilution water
14. Dilution factor
15. Number of dilutions3
16. Effect measured
17. Test acceptability
Static, non-renewal
25 ppt + 2 ppt
25°C + 1°C
Ambient laboratory
illumination
16 hr light, 8 hr dark
250-1000 ml
Minimum 200ml/replicate
7-21 days
10 (not to exceed loading
limits)
20
None
Natural seawater, synthetic
saltwater, or deionized water
mixed with hypersaline brine
0.5
5 plus a control. An
additional dilution at the
permitted effluent
concentration (% effluent) is
required if it is not included
in the dilution series.
Mortality-no movement on
gentle prodding.
90% or greater survival of
test organisms in control
solution.
(July 1, 1990)
65
-------
18. Sampling requirements
19. Sample volume required
For on-site tests, samples
must be used within 24 hours
of the time they are removed
from the sampling device.
Off-site test samples must be
used within 48 hours of
collection.
Minimum 4 liters
Footnotes;
1. Adapted from EPA/600/4-85/013.
2. If dissolved oxygen falls below 40% saturation, aerate at
rate of less than 100 bubbles/min. Routine DO checks
recommended.
3. When receiving water is used for dilution an additional
control made up of standard dilution water (0% effluent) is
required.
(July 1, 1990)
66
-------
VIII. CHEMICAL ANALYSIS
The following chemical analyses shall be performed for each
sampling event.
Minimum
Detection
Parameter Effluent Diluent Limit (ma/L)
pH xx
Specific Conductance x x
Salinity x x PPT(o/oo)
Total Residual Oxidants x x 0.02
Total Solids and Suspended Solids x x
Ammonia x x 0.1
Total Organic Carbon x x 0.5
Total Metals
Cd x 0.01.
Cr, Ni x 0.05
Pb, Zn, Cu x 0.01
Al x 0.02
Superscript;
1 Total Residual Oxidants
Methods: either of the following methods from the 16th
Edition of the APHA (1985) Standard Methods for the
Examination of Water and Wastewater must be used for these
analyses:
Method 408-C (Amperometric Titration Method)-the
preferred method;
Method 408-D (Ferrous Titration Method).
IX. TOXICITY TEST REPORT
The following must be reported:
Description of sample collection procedures, site
description;
Names of individuals collecting and transporting samples,
times and dates of sample collection and analysis; and
General description of tests: age of test organisms,
origin, dates and results of standard toxicant tests; light
and temperature regime; other information on test conditions
if different than procedures recommended.
(July 1, 1990)
67
-------
Toxicity test data shall include the following:
Survival for each concentration and replication at time 24,
and 48 hours.
LC50 and 95% confidence limits shall be calculated using one
of the following methods in order of preference Probit,
Trimmed Spearman Karber, Moving Average Angle, or the
graphical method. All printouts (along with the name of the
program, the date, and the author(s)) and graphical displays
must be submitted. When data is analyzed by hand,
worksheets should be submitted.
The Probit, Trimmed Spearman Karber, and Moving Average
Angle methods of analyses can only be used when mortality of
some of the test organisms are observed in at least two of
the (% effluent) concentrations tested (i.e. partial
mortality). If a test results in a 100% survival and
100% mortality in adjacent treatments ("all or nothing"
effect), a LC50 may be estimated using the graphical method.
All chemical data/physical generated (include detection
limits).
Raw data and bench sheets.
Any other observations or test conditions affecting test
outcome.
X. REPORTING
Signed copies of the toxicity testing reports shall be submitted
as required by of Part I of the permit.
(July 1, 1990) 68
-------
ATTACHMENT F
Mysid Shrimp (Mysidopsis bahia) 96 Hour Acute Test
(July 1, 1990) 69
-------
ATTACHMENT F
Acute Toxicity Test Procedure and Protocol:
o Mysid Shrimp (Mysidopaia bahia) definitive 96 hour test.
I. GENERAL REQUIREMENTS
The permittee shall conduct acceptable toxicity tests in
accordance with the appropriate test protocol described Below.
The permittee shall collect discharge samples and perform the
toxicity tests that are required by Part I of the NPDES permit.
Acute toxicity data shall be reported as outlined in Section
VIII.
II. TEST FREQUENCY AND SAMPLING REQUIREMENTS
See Part I of the NPDES permit for sampling location, sample
type, test frequency, test species, and test date(s)
requirements. Chain of Custody information should be provided
for each sample tested.
A sampling event is defined as a single discharge (composite or
grab) sample.
III. METHODS
Methods to follow are those recommended by EPA in:
Peltier, W., and Weber, C.I., 1985. Methods for Measuring the
Acute Toxicitv of Effluents to Freshwater and Marine Organisms.
Third Edition. Office of Research and Development, Cincinnati,
OH. EPA/600/4-85/013.
Any exceptions are stated herein.
IV. SAMPLE COLLECTION
A discharge sample shall be collected. Aliquots shall be split
from the sample(s), containerized and preserved (as per 40 CFR
Part 136) for the chemical and physical analyses. The remaining
sample shall be dechlorinated (if necessary) in the laboratory
using sodium thiosulfate for subsequent toxicity testing. Grab
samples must be used for pH, temperature, and total residual
oxidants (as per 40 CFR Part 122.21).
The Methods for Aquatic Toxicity Identification Evaluations
(Phase I) EPA/600/3-88/034, Section 8.7, provides detailed
information regarding the use of sodium thiosulfate (i.e.
dechlorination).
All samples held overnight shall be refrigerated at 4°C.
(July 1, 1990) 71
-------
V. DILUTION WATER
Dilution water used for acute toxicity analysis shall be
collected at a point away from the discharge which is free frora
toxicity or other sources of contamination. When using receiving
water as the dilution water an additional control (0% effluent),
made up from a laboratory water of known quality, will also be
run.
If the receiving water diluent is found to be, or suspected to be
toxic or unreliable, an alternate or standard dilution water of
known quality with a salinity, pH, conductivity, and total
suspended solids similar to that of the receiving water may be
substituted AFTER RECEIVING WRITTEN APPROVAL FROM THE PERMIT
ISSUING AGENCY(S). It may prove beneficial to the permittee to
have the proposed dilution water source screened for suitability
prior to toxicity testing. For further information see
Section 6, page 22 of EPA/600/4-85/013.
(July 1, 1990) 72
-------
VI. REGION I RECOMMENDED EFFLUENT TOXICITY TEST CONDITIONS FOR
THE MYSID fMvsidopsis bahia) 96 HOUR TEST1
1. Test type
2. Salinity
3. Temperature (°C)
4. Light quality
5. Photoperiod
6. Test chamber size
7. Test solution volume
8. Renewal of test solutions
9. Age of test organisms
10. No. Mysids per test chamber
11. No. of replicate test chambers
per treatment
12. Total no. Mysids per test
concentration
13. Feeding regime
14. Aeration3
15. Dilution water
brine.
16. Dilution factor
17. Number of dilutions*
Static, renewal2
25 ppt ± 10 percent for all
dilutions
25°C
Ambient laboratory
illumination
16 hour light, 8 hour dark
250 ml
200 ml
Daily
1-5 days
10
18. Effect measured
20
Light feeding (2 drops con-
centrated brine shrimp
nauplii, approx 100 nauplii/
mysid) twice daily
None
Natural seawater, synthetic
salt water, or deionized
water mixed with hypersaline
0.5
5 plus a control. An
additional dilution at the
permitted effluent
concentration (% effluent) is
required if it is not included
in the dilution series.
Mortality - no movement of
body or appendages on gentle
prodding
(July 1, 1990)
73
-------
19. Test acceptability
20. Sampling requirements
21. Sample volume required
90% or greater survival of
test organisms in control
solution
For on-site tests, samples are
collected daily and used
within 24 hours of the time
they are removed from the
sampling device. For off-
site tests, samples must first
be used within 48 hours of
collection.
Minimum 4 liters
Footnotes!
1,
2,
Adapted from EPA/600/4-85/013.
If the 96 hour mysid test is being performed concurrently
with chronic toxicity test(s), samples shall be collected on
days 0 and 3 of the 4-day exposure period. Renewal of test
concentrations for toxicity testing shall be conducted with
the most recently collected discharge sample. The initial
(day 0) sample will be analyzed chemically. Day 3 sample
will be held until test completion. If the renewal (day 3)
sample is of sufficient potency to cause lethality to 50
percent or more test organisms in any of the dilutions, then
a chemical analysis shall be performed on this sample as
well.
If dissolved oxygen falls below 40% saturation, aerate at
rate of less than 100 bubbles/min. Routine DO checks are
recommended.
When receiving water is used for dilution an additional
control made up of standard dilution water (0% effluent) is
required.
(July 1, 1990)
74
-------
VII. CHEMICAL ANALYSIS
The following chemical analyses shall be performed for'each
sampling event.
Minimum
Detection
Parameter Effluent Diluent Limit(mq/L)
PH
Specific Conductance
Salinity
Total Residual Oxidants
Total Solids and Suspended Solids
Ammonia
Total Organic Carbon
Total Metals
Cd
Cr, Ni
Pb, Zn, Cu
Al
X
X
X
X
X
X
X
X
X
X
X
X
X
X
• — •
PPT(o/oo)
0.02
0.1
0.5
X
X
X
X
0.01
0.05
0.01
0.02
In addition, the following chemical analyses shall be performed
as part of each daily renewal procedure on each dilution and the
controls.
Beginning of 24-hr
Exposure Period
End of 24-hr
Exposure Period
Parameter
Dissolved Oxygen . x x
Temperature x
pH x
Specific Conductance x
Superscript:
1 Total Residual Oxidants
Methods: either of the following methods from the 16th
Edition of the APHA (1985) Standard Methods for the
Examination of Water and Wastewater must be used for these
analyses:
Method 408-C (Amperometric Titration Method)-the
preferred method;
Method 408-D (Ferrous Titration Method).
(July 1, 1990)
75
-------
VIII. TOXICITY TEST REPORT
The following must be reported:
Description of sample collection procedures, site
description;
Names of individuals collecting and transporting samples,
times and dates of sample collection and analysis; and
General description of tests: age of test organisms,
origin, dates and results of standard toxicant tests; light
and temperature regime; other information on test conditions
if different than procedures recommended.
Toxicity test data shall include the following:
Survival for each concentration and replication at time 24,
48, 72, and 96 hours.
LC50 and 95% confidence limits shall be calculated using one
of the following methods in order of preference Probit,
Trimmed Spearman Karber, Moving Average Angle, or the
graphical method. All printouts (along with the name of the
program, the date, and the author(s)) and graphical displays
must be submitted. When data is analyzed by hand,
worksheets should be submitted.
The Probit, Trimmed Spearman Karber, and Moving Average
Angle methods of analyses can only be used when mortality of
some of the test organisms are observed in at least two of
the (% effluent) concentrations tested (i.e. partial
mortality). If a test results in a 100% survival and
100% mortality in adjacent treatments ("all or nothing"
effect), a LC50 may be estimated using the graphical method.
All chemical/physical data generated (include detection
limits).
Raw data and bench sheets.
Any observations and test conditions which affected the
outcome of testing.
IX. REPORTING
Signed copies of the toxicity testing reports shall be submitted
as required by of Part I of the permit.
(July 1, 1990) 76
-------
ATTACHMENT G
Sheepshead Minnow (Cvorinodon variecratual
Survival and Growth Test
(July 1, 1990) 77
-------
ATTACHMENT 6
Chronic Toxicity Test Procedure and Protocol:
o Sheepshead Minnow (Cvprinodon varieoatus) growth and
survival test.
I. GENERAL REQUIREMENTS
The permittee shall conduct acceptable toxicity tests in
accordance with the appropriate test protocols described below.
The permittee must collect discharge samples and perform the
toxicity tests that are required by Part I of the NPDES permit.
Chronic toxicity data shall be reported as outlined in Section
VIII.
II. TEST FREQUENCY AND SAMPLING REQUIREMENTS
See Part I of the NPDES permit for sampling location, sample
type, test frequency, test species, and test date(s)
requirements. Chain of Custody information should be provided
for each sample tested.
A chronic toxicity sampling event is defined as three discharge
(composite or grab) samples collected over the seven-day period
(see Section IV).
III. METHODS
Methods to follow are those recommended by EPA in:
Weber, C.I., et al, 1988. Short Term Methods for Estimating the
Chronic Toxicity of Effluents and Receiving Waters To Marine and
Estuarine Organisms. Office of Research and Development,
Cincinnati, OH. EPA/600/4-87/028.
Any exceptions are stated herein.
IV. SAMPLE COLLECTION
For each sampling event, three discharge samples shall be
collected over a 7-day exposure period. An initial sample (day
0) is used to start the test. The additional two samples are
collected for use at the start of day 3 and 5. Renewal of test
concentrations is conducted daily with the most recently
collected discharge sample. The initial (day 0) sample will be
analyzed chemically. Day 3 and 5 samples will be held until test
completion. If either the day 3 or 5 renewal sample is of
sufficient potency to cause lethality to 50 percent or more test
organisms in any of the dilutions, then a chemical analysis shall
be performed on the appropriate sample(s) as well.
Aliquots shall be split from the sample, containerized and
preserved (as per 40 CFR Part 136) for the chemical and physical
analyses. The remaining sample shall be dechlorinated (if
necessary) in the laboratory using sodium thiosulfate for •
subsequent toxicity testing. Grab samples must be used for pH,
(July 1, 1990) 79
-------
temperature, and total residual oxidants (as per 40 CFR Part
122.21).
The Methods for Aquatic Toxicity Identification Evaluations
fPhase I). EPA/600/3-88/034, Section 8.7, provides detailed
information regarding the use of sodium thiosulfate (i.e.
dechlorination).
All samples held overnight shall be refrigerated at 4°C.
V. DILUTION WATER
Dilution water used for toxicity analysis shall be collected at a
point away from the discharge which is free from toxicity or
other sources of contamination. When using receiving water as
the dilution water an additional control (0% effluent), made up
from a laboratory water of known quality, will also be run.
If the receiving water diluent is found to be, or suspected to be
toxic or unreliable, an alternate or standard dilution water of
known quality with a salinity, pH, conductivity, and total
suspended solids similar to that of the receiving water may be
substituted AFTER RECEIVING WRITTEN APPROVAL FROM THE PERMIT
ISSUING AGENCY(S). It may prove beneficial to the permittee to
have the proposed dilution water source screened for suitability
prior to toxicity testing. For further information see
Section 7, page 19 of EPA/600/4-87/028.
Dilution water used for marine chronic toxicity shall be of
sufficient quality to meet minimum acceptability of test results
(see Section VI).
(July 1, 1990) 80
-------
VI. REGION I RECOMMENDED TEST CONDITIONS FOR THE SHEEPSHEAD
MINNOW fCvprinodon varieaatus) LARVAL GROWTH AND SURVIVAL
TEST1
1. Test type
2. Salinity
3. Temperature
4. Light quality
5. Light intensity
6. Photoperiod
7. Test vessel size
8. Test solution volume
9. Renewal of test solutions
10. Age of test organisms
11. Larvae/test chamber
12. Number of replicate chambers
per treatment
13. Source of food
14. Feeding regime
15. Cleaning
16. Aeration2
17. Dilution water
Static, renewal
20 o/oo TO 30 o/oo ± 2-'o/o.o
25 ± 2°C
Ambient laboratory light
10-20 uE/m2/s, or 50-100 ft-c
(Ambient Laboratory Levels)
14 hr light, 10 hr darkness
300 - 1000 ml beakers or
equivalent
250 - 750 ml/replicate
(loading and DO restrictions
must be met)
Daily using most recently
collected sample.
Newly hatched larvae (less
than 24 hr old
15 (minimum of 10)
4 (minimum of 3)
Newly hatched Artemia nauplii
less than 24 hr old
Feed once a day 0.10 g wet wt
Artemia nauplii per replicate
on days 0-2; feed 0.15 g wet
wt Artemia nauplii per
replicate on days 3-6
Siphon daily, immediately
before test solution renewal
None
Uncontaminated source of
natural seawater; or
hypersaline brine; or
artificial seawater mixed with
deionized water
(July 1, 1990)
81
-------
18. Effluent concentrations
19. Dilution factor
20. Test duration
21. Effects measured
22. Acceptability of test
23. Sampling requirements
5 and a control. An
additional effluent
concentration (% effluent) is
required.
0.5
7 days
Survival and growth (weight)
The average survival of
control larvae is a minimum of
80%, and the average dry wt of
unpreserved control larvae is
a minimum of 0.6 mg, or the
average dry wt of preserved
control larvae is a minimum of
0.50 mg.
For on-site tests, samples are
collected daily and*used
within 24 hours of the time
they are removed from the
sampling device. For off-
site tests, samples must be
first used within 48 hours of
collection.
Footnotes:
Adapted from EPA/600/4-87/028.
If DO falls below 60% of saturation, aerate all chambers at
a rate of less than 100 bubbles/min. Routine DO checks are
recommended.
When receiving water is used for dilution an additional
control made up of standard dilution water (0% effluent) is
required.
(July 1, 1990)
82
-------
VII. CHEMICAL ANALYSIS
The following chemical analyses shall be performed for each
sampling event.
Minimum
Detection
Parameter Effluent Diluent Limitfma/Ll
PH
Specific Conductance
Salinity
Total Residual Oxidants1
Total Solids and Suspended Solids
Ammonia
Total Organic Carbon
Total Metals
Cd
Cr, Ni
Pb, Zn, Cu
Al
X
X
X
X
ids x
X
X
X
X
X
X
X
X
X
• .
PPT(o/oo)
0.02
0.1
0.5
X
X
X
X
0.01
0.05
0.01
0.02
In addition, the following chemical analyses shall be performed
as part of each daily renewal procedure on each dilution and the
controls.
Parameter
Dissolved Oxygen
Temperature
PH
Specific Conductance
Superscript:
Beginning of 24-hr
Exposure Period
End of 24-hr
Exposure Period
x
x
X
X
1
Total Residual Oxidants
Methods: either of the following methods from the 16th
Edition of the APHA (1985) Standard Methods for the
Examination of Water and Wastewater must be used for these
analyses:
Method 408-C (Amperometric Titration Method)-the
preferred method;
Method 408-D (Ferrous Titrimetric Method).
(July 1, 1990)
83
Printed on Recycled Paper
-------
VIII. TOXICITY TEST REPORT ELEMENTS
A report of results will include the following:
Description of sample collection procedures, site
description;
Names of individuals collecting and transporting samples,
times and dates of sample collection and analysis; and
General description of tests: age of test organisms,
origin, dates and results of standard toxicant tests; light
and temperature regime; other information on test conditions
if different than procedures recommended.
Toxicity test data shall include the following:
Daily survival of test organisms in the controls and all
replicates in each dilution if applicable.
Chronic test data shall undergo hypothesis testing to
determine if the distribution of results is normal using the
Shapiro-wilks test. The variance must also be tested for
homogeneity using Bartlett's Test. Then the endpoint
estimates, NOEC and LOEC must be determined using Dunnett's
Procedure, Bonferroni's T-Test, Steel's Many-One Rank Test,
or Wilcoxon Rank Sum Test. The choice of test depends on
the number of replicates and whether the variance is
homogeneous or not. See EPA/600/4-87/028 for details. All
printouts and graphical displays must be submitted.
C-NOEC: Chronic No Observed Effect Concentration.
LOEC: Lowest Observed Effect Concentration.
MATC: Maximum Allowable Toxicant Concentration.
All chemical/physical data generated (include detection
limits).
Raw data and bench sheets. (See sample data sheets pp. 85-
88.)
Any other observations and test conditions that may have
affected the outcome of the test.
IX. REPORTING
Signed copies of the toxicity testing reports shall be submitted
as required by Part I of the permit.
(July 1, 1990) 84
-------
Data forms for sheepshead minnow larval survival and growth test
Daily record of larval survival and test.conditions.'
Test Dates:
Type Effluent:
.Species:
Field
Lab
Test
c
*-•
*<
VO
VO
O
Effluent Tested:
CONCENTRATION:
REPLICATE:
DAYS
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5
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CONCENTRATION:
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6
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REPLICATE
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2
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4
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6
MfANWdGHI
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CONCENTRATION:
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i i i i i~m
COMMENTS:
Adapted from: Hughes, Heber. SchiM»e1, and Berry, 1987.
-------
Test Dates
Type Effluent
. Species
Field
Lab
Test
£ Effluent Tested
CONCENTRATION
REPLICATE
DAYS
nivf
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Itl
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r J
oo
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09
O\
i r
COMMENTS
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Data forms for sheepshead minnow larval survival
and growth test. Dry weights of larvae. <
Test Dates:
Species:
Pan
*
Cone.
&
Rep
Initial
Wt
(mg)
Final
Wt
(mg)
Diff
(mg)
Larvae
Av Wt/1
Larvae
(mg)
Adapted from: Hughes, Heber, Schimmel, and Berry, 1987.
(July 1, 1990)
87
Printed on Recycled Paper
-------
Data forms for sheepshead minnow larval survival
and growth test. Summary of test results.'
Test Dates
Effluent Tested
Species
TREATMENT
fLIVE
LARVAE
SURVIVAL
(%)
MEAN DRY WT /
LARVAE (mg)
±SD
SIGNIF OIFF
FROM CONTROL
(0)
MEAN
TEMPERATURE
(OC)
±SD
MEAN SALINITY
o/oo
±SO
AV. DISSOLVED
OXYGEN
(mg.'L) ± S D
COMMENTS:
(July 1, 1990)
88
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ATTACHMENT H
Chronic (and Modified Acute) Toxicity Test Procedure and
Protocol:
o Inland Silverside (Menidia beryllina) growth and survival
test.
I. GENERAL REQUIREMENTS
The permittee shall conduct acceptable toxicity tests in
accordance with the appropriate test protocols described below.
The permittee must collect discharge samples and perform the
toxicity tests that are required by Part I of the NPDES permit.
Chronic toxicity data shall be reported as outlined in Section
VIII.
II. TEST FREQUENCY AND SAMPLING REQUIREMENTS
See Part I of the NPDES permit for sampling location, sample
type, test frequency, test species, and test date(s)
requirements. Chain of Custody information should be provided
for each sample tested.
A chronic toxicity sampling event is defined as three discharge
(composite or grab) samples collected over the seven-day period
(see Section IV).
III. METHODS
Methods to follow are those recommended by EPA in:
Weber, C.I., et al, 1988. Short Term Methods for Estimating the
Chronic Toxicity of Effluents and Receiving Waters To Marine and
Estuarine Organisms. Office of Research and Development,
Cincinnati, OH. EPA/600/4-87/028.
Any exceptions are stated herein.
IV. SAMPLE COLLECTION
For each sampling event, three discharge samples shall be
collected over a 7-day exposure period. An initial sample (day
0) is used to start the test. The additional two samples are
collected for use at the start of day 3 and 5. Renewal of test
concentrations is conducted daily with the most recently
collected discharge sample. The initial (day 0) sample will be
analyzed chemically. Day 3 and 5 samples will be held until test
completion. If either the day 3 or 5 renewal sample is of
sufficient potency to cause lethality to 50 percent or more test
organisms in any of the dilutions, then a chemical analysis shall
be performed on the appropriate sample(s) as well.
(July 1991) 1
-------
Aliquots shall be split from the sample, containerized and
preserved (as per 40 CFR Part 136) for the chemical and physical
analyses. The remaining sample shall be dechlorinated (if
necessary) in the laboratory using sodium thiosulfate for
subsequent toxicity testing. Grab samples must be used for pH,
temperature, and total residual oxidants (as per 40 CFR Part
122.21).
The Methods for Aquatic Toxicity Identification Evaluations
(Phase I). EPA/600/3-88/034, Section 8.7, provides detailed
information regarding the use of sodium thiosulfate (i.e.
dechlorination).
All samples held overnight shall be refrigerated at 4°C.
V. DILUTION WATER
Dilution water used for toxicity analysis shall be collected at a
point away from the discharge which is free from toxicity or
other sources of contamination. When using receiving water as
the dilution water an additional control (0% effluent), made up
from a laboratory water of known quality, will also be run.
If the receiving water diluent is found to be, or suspected to be
toxic or unreliable, an alternate or standard dilution water of
known quality with a salinity, pH, conductivity, and total
suspended solids similar to that of the receiving water may be
substituted AFTER RECEIVING WRITTEN APPROVAL FROM THE PERMIT
ISSUING AGENCY(S). It may prove beneficial to the permittee to
have the proposed dilution water source screened for suitability
prior to toxicity testing. For further information see
Section 7, page 19 of EPA/600/4-87/028.
Dilution water used for marine chronic toxicity shall be of
sufficient quality to meet minimum acceptability of test results
(see Section VI).
(July 1991)
-------
VI. REGION I RECOMMENDED TEST CONDITIONS FOR THE INLAND
SILVERSIDE (Menidia bervllinal GROWTH AND SURVIVAL TEST1
1. Test type
2. Salinity
3. Temperature
4. Light quality
5. Light intensity
6. Photoperiod
7. Test vessel size
8. Test solution volume
9. Renewal of test solutions
10. Age of test organisms
11. Larvae/test chamber
12. Number of replicate chambers
13. Source of food
14. Feeding regime
15,
16,
Cleaning
Aeration2
Static, renewal
5 o/oo TO 32 o/oo + 2 o/oo
25 ± 2°C
Ambient laboratory light
10-20 uE/m2/s, or 50-100 ft-C
(Ambient Laboratory Levels)
16 hr light, 8 hr darkness
300 - 1000 ml beakers or
equivalent (glass test
chambers should be used)
250 ml minimum (loading and DO
restrictions must be met)
Daily using most recently
collected sample.
Seven to eleven days post
hatch.
15 (minimum of 10)
4 (minimum of 3)
per treatment
Newly hatched Artemia nauplii
less than 24 hr old
Feed once a day 0.10 g wet wt
Artemia nauplii per replicate
on days 0-2; feed 0.15 g wet
wt Artemia nauplii per
replicate on days 3-6
Siphon daily, immediately
before test solution renewal
and feeding
None
(July 1991)
-------
17. Dilution water
18. Effluent concentrations3
19. Dilution factor
20. Test duration
21. Effects measured
22. Acceptability of test
23. Sampling requirements
24. Sample Volume Required
Uncontaminated source of
natural seawater; or
deionized water mixed with
hypersaline brine or
equivalent artificial
seawater.
5 and a control. An
additional effluent
concentration (% effluent) is
required.
0.5
7 days
Survival and growth (weight)
The average survival of
control larvae is a minimum of
80%, and the average dry wt of
unpreserved control larvae is
a minimum of 0.5 mg, or the
average dry wt of preserved
control larvae is a minimum of
0.43 mg.
For on-site tests, samples are
collected daily and used
within 24 hours of the time
they are removed from the
sampling device. For off-
site tests, samples must be
first used within 48 hours of
collection.
Minimum of 5 liters.
Footnotes;
1
2
Adapted from EPA/600/4-87/028.
If DO falls below 60% of saturation, aerate all chambers at
a rate of less than 100 bubbles/min. Routine DO checks are
recommended.
When receiving water is used for dilution an additional
control made up of standard dilution water (0% effluent) is
required.
(July 1991)
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VII. CHEMICAL ANALYSIS
The following chemical analyses shall be performed for each
sampling event.
Minimum
Detection
Parameter Effluent Diluent Limitfmg/Ll
pH xx
Specific Conductance x x
Salinity x x PPT(o/oo)
Total Residual Oxidants x x 0.02
Total Solids and Suspended Solids x x
Ammonia x x 0.1
Total Organic Carbon x x 0.5
Total Metals
Cd x 0.01
Cr, Ni x 0.05
Pb, Zn, Cu x 0.01
Al x 0.02
In addition, the following chemical analyses shall be performed
as part of each daily renewal procedure on each dilution and the
controls.
Beginning of 24-hr End of 24-hr
Parameter Exposure Period Exposure Period
Dissolved Oxygen x x
Temperature x
pH x
Specific Conductance x
Superscript;
1 Total Residual Oxidants
Methods: either of the following methods from the 16th
Edition of the APHA (1985) Standard Methods for the
Examination of Water and Wastewater must be used for these
analyses:
Method 408-C (Amperometric Titration Method)-the
preferred method;
Method 408-D (Ferrous Titrimetric Method).
(July 1991)
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VIII. TOXICITY TEST REPORT ELEMENTS
A report of results will include the following:
Description of sample collection procedures, site
description;
Names of individuals collecting and transporting samples,
times and dates of sample collection and analysis; and
General description of tests: age of test organisms,
origin, dates and results of standard toxicant tests; light
and temperature regime; other information on test conditions
if different than procedures recommended.
Toxicity test data shall include the following:
Chronic
Daily survival of test organisms in the controls and all
replicates in each dilution.
Chronic test data shall undergo hypothesis testing to
determine if the distribution of results is normal using the
Shapiro-Wilks test. The variance must also be tested for
homogeneity using Bartlett's Test. Then the endpoint
estimates, NOEC and LOEC must be determined using Dunnett's
Procedure, Bonferroni's T-Test, Steel's Many-One Rank Test,
or Wilcoxon Rank Sum Test with Bonferroni adjustment. The
choice of test depends on the number of replicates and
whether the variance is homogeneous or not. See
EPA/600/4-87/028 for details. All printouts and graphical
displays must be submitted.
C-NOEC: Chronic No Observed Effect Concentration.
LOEC: Lowest Observed Effect Concentration.
- MATC: Maximum Allowable Toxicant Concentration.
All chemical/physical data generated (include detection
limits).
Raw data and bench sheets. (See sample data sheets pp. 8-
11.)
Any other observations and test conditions that may have
affected the outcome of the test.
Acute
Survival for each concentration and replication at time 24,
and 48 hours.
LC50 and 95% confidence limits shall be calculated using one
of the following methods in order of preference Probit,
Trimmed Spearman Karber, Moving Average Angle, or the
graphical method. All printouts (along with the name of the
program, the date, and the author(s)) and graphical displays
must be submitted. When data is analyzed by hand,
worksheets should be submitted.
The Probit, Trimmed Spearman Karber, and Moving Average
Angle methods of analyses can only be used when mortality of
some of the test organisms are observed in at least two of
the (% effluent) concentrations tested (i.e. partial
mortality). If a test results in a 100% survival and
100% mortality in adjacent treatments ("all or nothing"
effect), a LC50 may be estimated using the graphical method.
(July 1991) 6
-------
All chemical data/physical generated (include detection
limits).
Raw data and bench sheets.
Any other observations or test conditions affecting test
outcome.
IX. REPORTING
Signed copies of the toxicity testing reports shall be submitted
as required by Part I of the permit.
Any exceptions are stated herein.
(July 1991)
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C-i
c
vo
VO
Data forms for inland silver side larval survival and growth
test. Daily record of larval survival and test conditions.
Test Dales:
Type Effluent1
. Species
Field
Lab
Test
Effluent Tested:
CONCENTRATION:
REPLICATE
DAYS
•LIVE
LAHVAf
IEMP
rci
SALINITY
I'/.J
DO
Invj/l)
•IARVAE'
Dflvwn
0
1
2
3
4
5
MEAN WEIGHT/
LARVAE |mg) 1 S D
6
7
REPLICATE
0
1
•LARVAE
ORVWT
2
3
4
5
6
MIAN WEIGMI
LARVAE |mg| • S 0
7
REPLICATE
O
1
•LARVAE
ORVWT
2
3
4
5
6
7
REPLICATE
0
1
•LARVAE
ORVWI
2
3
4
5
6
MEAN WEIGHT
IARVAE Imgl ' SO
7
CONCENTRATION:
•LIVE
LARVAE
TEMP
I'CI
SALINITY
T/.J
OO
Ing/I)
•LARVAE/
DRV WT
MEAN WEIGHT/
LARVAE Imgl 1 S O
•LARVAE
ORVWT
MEAN WEIGHT
LARVAE Imgl • S D
n ARVAE
ORVWT
•LARVAE
ORVWT
1
MEAN WEIGHT •
LARVAE Imgl I S O
CONCENTRATION:
•LIVE
IARVAI
TEMP
CCI
SAiiHirv
r -i
DO
|mg/l)
nARVAi/
ORV WT
MEAN WEIGHT/
LARVAE Imgl 1 S O
•LARVAE
ORVWT
ME AN WEIGHT
LARVAE |mg| • S D
•LARVAE
DRY WT
•LARVAE
ORVWI
ME AN WEIGMI/
t ARVAE |mg| I SO
00
Ig I I I I I
COMMENTS:
-------
(Co ^ued).
Test Dates:
Type Effluent:
Species:
Field
Lab
Test
Effluent Tested:
CONCENTRATION:
REPLICATE:
DAYS
HIVE
LARVAE
TEMP
I-CI
SALINITY
r/J
DO
img/l)
•LARVAE/
DRY Wl
0
1
2
3
4
5
ME AN WEIGHT/
LARVAE Imgl t S O
6
7
0
1
•LARVAE
DRV WT
REPLICATE:
2
3
4
5
6
MEAN WEIGHT
.ARVAE Imgl ' S D
7
REPLICATE:
0
1
•LARVAE
DRV WT
2
3
4
5
6
7
REPLICATE:
0
1
•LARVAE
DRV WT
2
3
4
S
6
MEAN WEIGH!
LARVAE |mg| ' S O
7
CONCENTRATION:
•live
LARVAE
TEMP
I'CI
SALINITY
I'/J
DO
|mg/l)
•LARVAE/
DRV WT
ME AN WEIGHT/
LARVAE |mg| ( S O
•LARVAE
DRY WT
MEAN WEIGHT •
LARVAE Imgl > S O
•LARVAE
DRV WT
•LARVAE
DRV WT
ME AN WEIGHT-
LARVAE Imgl I S D
CONCENTRATION:
•LIVE
LARVAE
TEMP
I*CI
SALINITY
r -i
DO
lmg/1)
•LARVAE''
DRV V+T
MEAN WEIGH!/
LARVAE Imgl t SO
•LARVAE
DRY Wl
MEAN WEIGHT.
LARVAE |mg| I S D
• LARVAE
DftV Wl
•LARVAE •
DRV WT
MEAN WEIGHT/
LARVAE |mg| 1 S D
VO
COMMENTS:
IIIME
IED
1
1
-------
Test Dates:
Data forms for inland silverside larval survival
and growth test. Dry weights of larvae.
Species:
Pan
#
Cone.
&
Rep.
Initial
Wt.
(mg)
Final
Wt.
(mg)
Diff.
(mg)
Larvae
Av. Wt./
Larvae
(mg)
(July 1991)
10
-------
Test Dates:
CXAMPL6" Data forms for inland silverside larval
survival and growth test. Summary of test
results.
Species:
Effluent Tested:
TREATMENT
#LIVE
LARVAE
SURVIVAL
(%)
MEAN DRY WT./
LARVAE (mg)
±S.D.
SIGNIF. DIFF.
FROM CONTROL
(0)
MEAN
TEMPERATURE
(oC)
±S.D.
MEAN SALINITY
0/00
±S.D.
AV. DISSOLVED
OXYGEN
(mg./L) ±S.D.
•
COMMENTS:
(July 1991)
11
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