Reporting and Testing Guidance for Biomonitoring

Permit Guidance 5 Biomonitoring Test Guidance July 98 Page 1

Permit

Guidance

5

Final

Reporting and Testing Guidance for Biomonitoring

Required by the Ohio Environmental Protection Agency

Rule reference: 40CFR Part 136.3;

OAC 3745-2-09; OAC 3745-33-07

Ohio EPA, Division of Surface Water

Revision 0, October 1991

Revision 1, July 1998

This internal guidance does not affect the requirements found in the referenced rule or statute.

Purpose

This document provides detailed instructions to Ohio EPA staff and other professionals who

collect and analyze water samples for biomonitoring (bioassays) required by NPDES permits in

Ohio.

Background

The manual is intended to provide detailed information on the most frequently used procedures,

methods, quality assurance practices and data reporting techniques for bioassay tests.

Procedure

The report is attached. Contact the office listed below for more information.

Related Policy or guidance

Ohio EPA. 1995. Manual of Ohio EPA Laboratory Standard Operating Procedures.

Volumes I, II, III and IV.

For more information contact:

Ohio EPA, Division of Surface Water

Industrial Permit Group Leader

(614) 644-2001

C:\Documents and Settings\rheitzma\My Documents\permit5.wpd

TABLE OF CONTENTS

Page No.

Introduction ..................................................................................................... 1

Section 1: Mandatory Requirements for NPDES Biomonitoring

A. Use of Approved Methods ........................................................................ 2

B. Standard Operating Procedures ................................................................. 3

Section 2: Requirements for Acute Toxicity Testing

A. Acute Toxicity as an Effluent Characteristic ............................................ 4

B. Test Organisms .......................................................................................... 4

C. Length of Acute Toxicity Tests ................................................................. 4

D. Type of Test .............................................................................................. 5

E. Sample Collection ...................................................................................... 6

F. Quality Assurance ...................................................................................... 7

G. Chemical Analysis ..................................................................................... 8

H. Reporting Requirements ............................................................................ 9

Section 3: Requirements for Chronic Toxicity Testing

A. Chronic Toxicity as an Effluent Characteristic ......................................... 14

B. Test Organisms .......................................................................................... 15

C. Length of Chronic Toxicity Tests .............................................................. 15

D. Type of Test ............................................................................................... 15

E. Sample Collection ...................................................................................... 17

F. Quality Assurance ...................................................................................... 18

G. Chemical Analysis ..................................................................................... 19

H. Reporting Requirements ............................................................................ 20

Section 4: Requirements for Instream Biosurveys

A. Function of Instream Biological Surveys .................................................. 26

B. Factors Examined in Conducting a Biosurvey .......................................... 26

C. Time Period for Performing Survey Work ................................................ 27

D. Type of Survey .......................................................................................... 27

E. Sample Collection ...................................................................................... 27

F. Study Plan Submission .............................................................................. 27

G. Sampling of Ambient Waters for Instream Biosurveys ............................ 28

H. Reporting Requirements ............................................................................ 28

I. Collection Permit ........................................................................................ 30

Section 5: NPDES Permit Application Requirements

A. Guidelines for Submittal of This Data ...................................................... 30

Attachment 1: Form 4500

Attachment 2: Example Form 4500 Showing Acute Toxicity Test Results

Attachment 3: Ohio EPA NPDES Biomonitoring Report Form, Acute Toxicity Test

Attachment 4: Example Form 4500 Showing Chronic Toxicity Test Results

Attachment 5: Ohio EPA NPDES Biomonitoring Report Form, Chronic Toxicity Test

Attachment 6: Biosurvey Sampling Information

1

REPORTING AND TESTING GUIDANCE FOR BIOMONITORING

REQUIRED BY OHIO ENVIRONMENTAL PROTECTION AGENCY

Introduction

This document has been prepared to provide guidance on the reporting and testing requirements

for biomonitoring conditions in a National Pollutant Discharge Elimination System (NPDES)

permit. It includes important changes to reporting procedures for biomonitoring, as well as

clarification of test procedures.

Biomonitoring is designed to evaluate the impact or potential impact of a wastewater discharge

on aquatic life using biological methods. Biomonitoring requirements may come in two basic

forms: 1) evaluating effluent toxicity through toxicity tests; and 2) evaluating the impact of an

effluent through assessment of the instream community.

Toxicity testing uses aquatic organisms to directly measure effluent toxicity and is the most

common form of biomonitoring included in NPDES permits. Typically, NPDES permits in Ohio

will require toxicity testing using fathead minnows (Pimephales promelas) and Ceriodaphnia

dubia, although Ohio EPA will consider the use of alternative test species. Prior approval by

Ohio EPA is required for use of alternative test species.

There are three methods of effluent toxicity testing, flow-through, static, and static renewal. A

flow-through toxicity test requires that the tested effluent be constantly pumped through the test

chamber. A static test requires that the sample used to initiate the test is the only one used for

the duration of the test. A static renewal test requires that the test solutions be renewed daily

using the original sample or additional samples collected during the testing period.

Toxicity tests may also measure different types of effects depending upon the duration and intent

of the test. Acute toxicity tests measure short-term, obvious effects. Chronic toxicity tests

measure longer-term, but potentially more subtle effects. The difference between these two

types of tests involve the duration of the test and the toxicity end points measured in the tests.

An acute toxicity test usually has a duration of 2 to 4 days, depending upon the test species. The

end points measured are death or atypical behavior or appearance. A chronic toxicity test may

last as long as 1 year or more. Early life stage chronic tests may last 28 to 30 days. Typically, a

chronic or sub-chronic toxicity test required by an NPDES permit is for the shorter term of 7

days. The end points measured are growth or reproductive effects, as well as death and/or

atypical behavior or appearance.

The second form of biomonitoring is the instream community assessment. It is a direct measure

of the structure and function of the aquatic community living in the receiving water. The

assessment is made by sampling the resident populations and comparing existing aquatic

2

populations to the criteria for minimally-impacted aquatic communities established in the Ohio

Water Quality Standards (WQS).

Ohio EPA may require some or all of the above testing requirements based upon factors that

apply to a particular facility. This document identifies the specific methods to be used in

conducting biomonitoring programs and provides guidance on how to fulfill reporting

requirements of the NPDES permit.

Section 1: Mandatory Requirements for NPDES Biomonitoring

A. Use of Approved Methods.

The use of Ohio EPA-approved methods is required for biomonitoring conducted pursuant

to the terms of an NPDES permit. Alternative methods may be used only after obtaining

prior approval from Ohio EPA and USEPA. In order to solicit approval for an alternative

method, the entity should submit a written request to:

Ohio EPA - Division of Surface Water

P.O. Box 1049

Columbus, Ohio 43216-1049

The following are approved test methodologies for biomonitoring being conducted as a

required of an NPDES permit

1. Approved Methods for Toxicity Testing:

The following documents contain methods that are approved for use in conducting

effluent or other toxicity testing:

a. 40 CFR 136.3 (Tables 1A and II).

Methods to Measure the Acute Toxicity of Effluent and Receiving Waters To

Freshwater, Estuarine and Marine Organisms

Short-Term Methods to Estimate the Chronic Toxicity of Effluents and

Receiving Waters to Freshwater Estuarine and Marine Organisms

b. Ohio EPA Quality Assurance Manual (current edition).

These documents can be obtained by contacting Ohio EPA-Division of

Environmental Services, Bioassay Section, P.O. Box 1049, Columbus, Ohio 43216-

1049.

3

2. Approved Methods for Instream Biological Assessment:

The following documents contain methods that are approved for use in conducting stream

surveys to demonstrate attainment of biological criteria established in the Ohio WQS (Ohio

Administrative Code (OAC) 3745-1-07. All of the following documents are required to be

utilized in the design, execution and reporting of biomonitoring conducted for comparison

with the biocriteria contained in OAC 3745-1-07:

a. Ohio EPA Surveillance Methods and Quality Assurance Manual (current edition).

b. Biological Criteria for the Protection of Aquatic Life: Volume I. The Role of

Biological Data in Water Quality Assessment, Ohio EPA, 1987.

c. Biological Criteria for the Protection of Aquatic Life: Volume II. Users Manual for

Biological Field Assessment of Ohio Surface Waters, Ohio EPA, 1987.

d. Addendum to Biological Criteria for the Protection of Aquatic Life: Volume II.

Users Manual for Biological Field Assessment of Ohio Surface Waters, Ohio EPA,

1989.

e. Biological Criteria for the Protection of Aquatic Life: Volume III. Standardized

Biological Field Sampling and Laboratory Methods for Assessing Fish and

Macroinvertebrate Communities, Ohio EPA, 1989.

f. The Qualitative Habitat Evaluation Index (QHEI): Rationale, Methods and

Application, Ohio EPA, 1989.

These documents can be obtained by contacting Ohio EPA-Division of Surface Water, P.O.

Box 1049, Columbus, Ohio 43216-1049.

B. Standard Operating Procedures.

Whether the permittee performs the biomonitoring work or retains a contract laboratory or

consulting firm to perform the necessary testing, it is necessary to submit an official

Standard Operating Procedure (SOP) to Ohio EPA. The SOP is a detailed explanation of

the actual techniques used to conduct tests required by NPDES permits. The submission of

an SOP will enable Ohio EPA to minimize the reporting requirements necessary for each

test result, as well as verify that approved test methodologies are being utilized.

If the permittee chooses to retain a contract laboratory, a previous SOP submittal will be

sufficient to fulfill this condition, so long as the SOP accurately reflects the laboratory’s

current operations. If an SOP has not been submitted, the permittee is responsible for

assuring the timely submittal of an SOP by the laboratory performing the testing. If an

4

SOP has previously been submitted, the permittee should notify Ohio EPA at the address

below, indicating the date and title of the submittal. Any necessity to deviate from the SOP

due to special case considerations should be noted as such on the test report. Ohio EPA

should be consulted prior to conducting the test if a reason for deviating from the SOP is

known to exist at that time. Ohio EPA reserves the right to comment on an SOP

concerning issues perceived to be in conflict with approved methods.

SOP’s should be submitted, in duplicate, to:

Permits Section

Ohio EPA-Division of Surface Water

P.O. Box 1049

Columbus, Ohio 43216-1049

Section 2: Requirements for Acute Toxicity Testing

This Section contains NPDES permit requirements for effluent toxicity testing designed to

measure acute toxicity. These requirements should be followed unless specifically modified by

the NPDES permit.

A. Acute Toxicity as an Effluent Characteristic.

Acute toxicity in an effluent toxicity test is measured as a short-term effect induced by

exposure to an effluent. The end points for an acute toxicity test are generally death of the

organism and/or atypical behavior or appearance (which is termed an effect). Summary

statistics, such as median lethal concentration (LC

50

) or percent morality, are determined

following completion of the test.

B. Test Organisms.

Test organisms used for toxicity testing required by NPDES permits will be Pimephales

promelas (fathead minnow) and Ceriodaphnia dubia. Additional test species may be

required based upon site-specific factors.

Alternative test species may be used for toxicity testing only with the prior approval of

Ohio EPA.

C. Length of Acute Toxicity Tests.

Acute toxicity tests will be for the durations specific for the following organisms:

5

1. Ceriodaphnia dubia: Acute toxicity tests using Ceriodaphnia dubia will be 48 hours in

duration. Test organism survival and observations of behavior and external

appearance shall be recorded every 24 hours at a minimum.

2. Pimephales promelas: Acute toxicity tests using fathead minnows will be 96 hours in

duration except as indicated in Section 2.D.1.a. Test organism survival and

observations of behavior and external appearance shall be recorded every 24 hours at

a minimum.

D. Type of Test.

Acute toxicity tests required by NPDES permits or Director’s Final Findings & Orders

(DFFO’s) will be static tests. Static renewal or flow-through tests may be performed only

as approved or required by Ohio EPA.

Acute toxicity tests may be categorized as either screening tests or definitive tests. A

screening test is a test in which the effluent and control solutions are evaluated at full

strength. This is an inexpensive test designed to indicate presence or absence of toxicity in

the effluent. A definitive test uses dilutions of the effluent to quantify toxicity. These two

types of tests are described further below:

1. Acute Screening Tests: Ohio EPA uses screening toxicity tests of different durations

depending upon the intended use of the toxicity data. These are described below:

a. General Screening Tests - These tests are performed by Ohio EPA or the

permittee to screen for toxicity in the effluent. The effluent and control

solutions are tested full strength, but the test durations are 48 hours for both

Pimephales promelas and Ceriodaphnia dubia.

b. Additive Screening Tests - These tests may be required to be conducted as a

condition of an Ohio EPA Administrative Order (DFFO’s) approving the use of

a cooling water additive. The effluent and control solutions are tested full

strength and test durations are 96 hours for Pimephales promelas and 48 hours

for Ceriodaphnia

dubia.

2. Acute Definitive Tests: A definitive test is designed to quantify the amount of

toxicity in an effluent. In order to accomplish this, the effluent is diluted to various

concentrations with one of the control waters. A minimum of 5 effluent

concentrations shall be used in a definite test. The typical dilutions used are 100, 50,

25, 12.5 and 6.25 percent by volume effluent.

There may be instances when it will be advantageous to use a different dilution

series. Selection of the appropriate dilution series is the responsibility of the

6

permittee. Ohio EPA staff will be available for consultation on the issue of

appropriate dilution series. However, the permittee, in conjunction with the testing

laboratory, may exercise professional judgement on the selection of appropriate

dilution series. When selecting a dilution series, the following should be considered:

a. Allowable Effluent Toxicity (AET) - AET is the permissible amount of toxicity

for a particular discharge. This value is determined by factoring the available

dilution in the receiving stream with the water quality criteria for toxicity as

well as the effects of any interactive discharges. Should permit limitations for

toxicity be established for a permittee, the AET is the amount of toxicity that

would be allowed. The method for determining the AET is established in OAC

3745-2-09. AET values are also given in NPDES fact sheets and wasteload

allocations. When selecting a dilution series, the relationship of the data

obtained through use of that series to the AET should be considered. The series

selected should be one which will yield data to determine if the AET has/has

not been exceeded, yet still identifies the LC

50

.

b. Toxicity in the Effluent - There may be instances where it is necessary to alter

the dilution series in order to better determine an LC

50

. This would be the case

in an effluent that is moderately acutely toxic. In order to give a better estimate

of the LC

50

, the dilution series has to be shifted to a higher set of dilutions.

Also, if effluent toxicity is consistently exhibited within a certain range, it may

be advisable to adjust the dilution series to focus in that area to better define the

LC

50

value.

For example, an effluent has an LC

50

of roughly 78 percent effluent and the

dilution series used in testing is 100, 50, 25, 12.5 and 6.25 percent volume by

effluent. Test results would often turn up as 100 percent morality in 100

percent effluent, and no effect in 50 percent effluent. If the effluent is not too

variable, the dilution series should be shifted upward to better characterize the

apparent LC

50

of 78 percent effluent. A dilution series of 100, 75, 50, 25 and

12.5 percent effluent by volume might be used in this instance, to better define

the LC

50

.

E. Sample Collection.

Effluent samples used to conduct the acute toxicity tests shall be collected as 24-hour

composite samples. If the effluent is chlorinated for disinfection purposes, the effluent

sample should be collected at a point prior to chlorination. The protocols in Section 1.A.1

should be consulted for the handling of a chlorinated sample if it is impossible to obtain a

sample prior to disinfection. However, if dechlorination is an integral part of the

disinfection system at the facility, the sample should be collected at the final outfall.

7

Unless specifically modified by the NPDES permit, an acute toxicity test of an effluent

requires that an upstream control and a near-field downstream sample be collected. The

upstream control sample is to be collected as a grab sample upstream from the zone of

effluent and receiving water interaction. Care should be taken to assure than any upstream

backflow of the effluent is taken into account when selecting the upstream sampling

location.

The near-field downstream sample is to be collected as a grab sample from within the

effluent plume in the immediate vicinity of the outfall. The near-field sample should be

collected in the middle of the effluent plume at a distance of 5 times the water depth at the

point of discharge, down current from the outfall. When water depth at the point of

discharge exceeds 4 feet, the near-field sample should be collected 20 feet (6 meters) down

current from the outfall.

The location of the near-field sample with respect to the effluent plume must be determined

and documented at the time of sampling using temperature measurements, conductivity

measurements, visual observation, a dye study, or other detailed techniques for delineating

the effluent plume.

Samples taken for toxicity testing purposes must be used within 36 hours after completion

of sample collection.

F. Quality Assurance.

1. Requirements for the Repeating of a Test:

There may be instances when poor survival or other adverse effects exhibited by

control organisms preclude the use of data from an effluent toxicity test and the test

must be repeated. The following conditions outline when a repeat of an acute toxicity

test is mandatory. Failure to repeat a toxicity test when necessary may result in

monitoring frequency violations of NPDES permit conditions.

A repeat of an acute toxicity test is mandatory when a combination of mortality and

adverse effects in both

receiving water and laboratory controls exceeds 10 percent of

a particular species. A repeat test is not necessary if there is 10 percent or less

affected in one of the two control waters. A repeat test is necessary only for the

species exhibiting unacceptable effects in the controls.

Failure to follow approved procedures may result in a requirement to repeat a toxicity

test. Any deviations from approved procedures should be explicitly described in the

report of the tests results.

2. Dilution Water Substitution:

8

If a combination of mortality and adverse effects in the upstream control sample

exceeds 10 percent for a particular test organism in a sample, an alternative dilution

and control water should be identified and used for subsequent tests. Acceptable

alternative dilution waters may be similar natural waters, rearing unit water,

reconstituted water, or dilute mineral water. An upstream receiving water sample

must still be collected and tested in subsequent tests, but shall not be used as diluent.

Failure to change the dilution water in subsequent tests, following a test with

excessive mortality in the receiving water control, may result in invalidation of

testing results and a requirement to repeat the test.

3. Number of Organisms:

At least 20 organisms of a particular species shall be used for each solution tested in

an acute toxicity test.

4. Test Temperature:

The approved methods for acute toxicity listed in Section 1.A.1 contain different test

temperatures for the species depending upon the purpose and type of tests to be

conducted. For data comparability and uniformity between acute and chronic tests, a

test temperature of 25

o

+ 1

o

C should be used in acute tests. The test temperature that

will be used should be listed in the SOP.

5. Feeding During Testing:

The U.S. EPA guidance manual for acute toxicity testing recommends feeding C.

dubia during an acute toxicity test if the solutions are renewed at 48-hours of

exposure. However, due to the fact that Ohio EPA is specifying static acute tests, we

recommend that the organisms should not be fed during the test. The pool of

organisms that are to be used for the tests should be fed while in holding immediately

prior to test initiation. A minimum amount of this water should be transferred to the

test solutions.

G. Chemical Analysis:

A sufficient volume of effluent shall be collected to allow aliquots for use in acute toxicity

tests and chemical analysis. Bioassay effluent sampling may be coordinated with other

permit sampling requires as appropriate to avoid duplication. The analyses detailed in the

currently effective Part I, Effluent Limitations and Monitoring Requirements tables in the

NPDES permit must be conducted for the effluent sample. In addition, alkalinity and

hardness (as CaCO

3

) should be measured. Chemical analysis must comply with Ohio EPA

accepted procedures.

9

H. Reporting Requirements:

1. Reporting Toxicity Testing Results on Monthly Operating Reports - NPDES permits

require that results of testing be reported on a form acceptable to Ohio EPA. The

results of toxicity tests required under Part I Permit Requirements shall be reported

on EPA Form 4500. A copy of Form 4500 is included as Attachment 1.

Results for final effluent toxicity shall be expressed as toxic units on Form 4500. An

acute toxic unit (TU

a

) is defined as:

TU

a

= 100

LC

50

Form 4500 should be received by Ohio EPA Central Office by the 15th day of the

month following the month in which the toxicity test samples were collected. It may

be necessary to obtain or provide the toxic unit results prior to receipt of the full

laboratory report in order to fulfill this requirement. The toxicity test result should be

recorded on the first day of sampling for the toxicity test.

For purposes of reporting on Form 4500 only, the following conventions should be

used to report effluent toxicity tests where an LC

50

cannot be identified:

Percent Mortality or Other Adverse

TU

a

Effect in Whole (100%) Effluent

0.9 45

0.8 40

0.7 35

0.6 30

0.5 25

0.4 20

0.3 15

0.2 10

AA (below detectable) <10

The above conventions should be used for reporting valid toxicity tests (i.e., tests that

satisfy the requirements of Section 2.F). If a test is not valid due to control mortality

or other problems, the code “AE” (analytical data not valid) should be used to report

the test results on Form 4500. The test should then be repeated as appropriate.

Reporting for instream stations shall be in percent of organisms affected. This

percentage should reflect all mortality and atypical behavior or appearance by the test

10

organisms. Results from upstream stations (those NPDES monitoring stations

numbered as 801, 802, etc.) should be reported as the percent affected from that

sample. In the event that an alternative control/dilution water is used, data from the

upstream ambient station (if required) should still be reported and the fact that an

alternative dilution water was used should be recorded in the comments section on

the form. Downstream stations (those NPDES monitoring stations numbered as 901,

902, etc.) should reflect the results of the near-field sample. If no mortality or effects

are observed in these solutions, record “AA” (below detectable) on the 4500 form.

See Attachment 2 for an example of a completed Form 4500 showing the results of an

acute toxicity test.

Timely submittal of these data allows for input of toxicity testing results into Ohio ‘s

mainframe computer systems.

2. Information Reported for Acute Toxicity Tests:

Ohio EPA has established two basic reporting formats for toxicity testing results,

depending upon whether the permittee has effective toxicity unit (TU

a

) limitations in

its NPDES permit.

a. Reporting for Permittees with Detailed Reporting Requirements - If the

permittee does not have effective toxic unit limitations in its permit, additional

information shall be submitted, as well as the necessary reporting on Form

4500. Reports containing the additional information for acute toxicity

monitoring requirements shall be submitted to the following address within 60

days of the initiation of the test:

Permits Section

Ohio EPA-Division of Surface Water

P.O. Box 1049

Columbus, Ohio 43216-1049

Reporting of acute toxicity test results shall be submitted on the “Ohio EPA

NPDES Biomonitoring Report Form” (see Attachment 3). The biomonitoring

report consists of the following five parts: 1) General Information; 2) Acute

Toxicity Test Sampling Data; 3) Toxicity Test Conditions; 4) Acute Toxicity

Test Results and 5) Additional Toxicity Test Information and

Conclusions/Comments. The following is guidance on completion of the five

portions of the biomonitoring report:

1. General Information - Much of the information in this part is self-

explanatory. However, additional details follow for some items:

11

o

Reporting Date is the date the report is submitted

o

SOP’s are the standard operating procedures required by the NPDES

permit and described in Section 1.B. An SOP need only be submitted

once for any particular testing laboratory; the SOP on file with Ohio

EPA must be accurate and current. If the testing laboratory has

previously submitted an SOP, mark “yes” and indicate the date the SOP

was submitted.

o

The Certification Statement and Signature form must be signed by a

responsible person in charge of the facility as defined by 40 CFR

122.22.

2. Acute Toxicity Test Sampling Data - Describe conditions relating to the

collection of samples to be tested. This part should be filled out as

necessary for each outfall that is tested. The information entered in this

part of the form is self-explanatory.

3. Toxicity Test Conditions - Describe the conditions during the toxicity test.

This part should be filled out for each species tested. Additional details

describing some of the information requested are listed below:

o

For Test Type and Duration, the test type would typically be static;

durations should be listed in hours.

o

The Light Quality states the type of lighting system and/or light

intensity, if available.

o

For Dilution and Primary Control Water, list the source of the dilution

and primary control water (e.g., receiving water, rearing unit water,

dilute mineral water, etc.).

o

The Secondary Control Water lists the source of the secondary control

water (e.g., hard reconstituted water, receiving water, dilute mineral

water, etc.).

o

For aeration, indicate whether or not the sample required aeration and if

aeration was required before or during the test.

4. Acute Toxicity Test Results - Describe the results of the acute toxicity

test. This part should be filled out for each species tested and for each

outfall tested. Much of the information that is requested is self-

explanatory. Additional details describing some of the information

requested are listed below:

12

o

Cumulative Percent Mortality is the cumulative total of dead organisms,

expressed as a percentage of the total test organisms for that solution

recorded every 24 hours. The result should be recorded on the line

corresponding to the solution that was tested.

o

Cumulative Percent Affected is the cumulative total of organisms

showing some adverse effect (e.g., death, disorientation, atypical

appearance or behavior), expressed as a percentage of the total test

organisms for that solution recorded every 24 hours. The result should

be recorded in the parentheses below the line showing percent mortality

at the time for that solution.

o

For Method(s) Used to Determine ..., list the graphical or statistical

methods used to derive the LC

50

, and their 95 percent confidence limits.

5. Additional Toxicity Test Information - This portion is used to present

information about the effluent plume during sampling, as well as any

conclusions that may be drawn from the data, and should be completed for

each species and outfall. Additional details on the information requested

are listed below:

o

For Method(s) Used to Verify Near-Field and/or Far-Field Sampling

Locations ..., complete this portion indicating the method used to verify

the location of the effluent plume during the sampling (e.g.,

conductivity, temperature, visual, etc.).

o

Conclusions/Comments should be completed for each species and

outfall tested as the permittee or testing laboratory as deemed

appropriate. The information listed under Additional Toxicity Test

information must be attached to the report.

b. Reporting for Permittees with Toxic Unit Limits - If the permittee has effective

toxic unit permit limitations, the only reporting required is on Form 4500,

provided that the SOP requirements (see Section 1.B) has been fulfilled. All

other data elements required by this Section (see items 1 through 21, below)

shall be retained by the permittee in accordance with Part III.7, Records

Retention, of the NPDES permit. Although records retention requirements

specify that records be maintained for 3 years, Ohio EPA recommends that

toxicity test information be retained for 5 years (or through the subsequent

permit renewal).

13

The following information is required to be retained as supporting information

for each test. This data will be used, if necessary, to assure the validity of data

submitted on Form 4500.

1. Name and address of the testing laboratory.

2. Name and address of the facility which is the source of the effluent tested.

3. Ohio EPA and NPDES permit numbers for the facility.

4. Receiving water tested and/or used.

5. Source of dilution water.

6. Date and time of sample collection.

7. Collector(s) name(s).

8. Type of toxicity test.

9. Test organisms used.

10. Test organism origin and acclimation process.

11. Number of organisms per container and per concentration.

12. Test container size, number per concentration, and depth of test solution.

13. Concentrations tested and volume.

14. Test temperature.

15. Results of chemical analyses.

16. Results of physicochemical measurements taken during the test.

17. Definition of adverse effects measured in the test (end points).

18. Number of organisms in each concentration showing the adverse effects at

specified times.

14

19. Median lethal concentrations (LC

50

) for each 24-hour interval during the

test, confidence limits for those values, and the methods used for the

calculations.

20. Exact details for the near-field sample location in relation to the outfall

and plume location along with the results from temperature, conductivity

or dye study used to confirm the effluent plume. If conducted, results of

the detailed mixing zone study shall be provided.

21. Any other relevant information.

Section 3: Requirements for Chronic Toxicity Testing

This Section discusses the details of NPDES permit requirements for effluent toxicity testing

designed to measure chronic toxicity. These requirements should be followed unless specifically

modified by the NPDES permit.

A. Chronic Toxicity as an Effluent Characteristic.

Chronic toxicity in an effluent toxicity test is measured as an effect or effects induced by a

relatively long-term exposure to an effluent. The end points for a chronic toxicity test are

growth or reproductive effects, as well as death of an organism and atypical appearance or

behavior. End results of chronic toxicity tests are defined by the Lowest Observed Effect

Concentration (LOEC), the No Observed Effect Concentration (NOEC), and the Inhibition

Concentration (IC). The IC is the concentration that would cause a given percent reduction

in a non-quintal biological measurement (e.g., the IC25 would cause a 25 percent reduction

in mean young Ceriodaphnia dubia reproduction or in Pimephales promelas growth, and

the IC50 would cause a 50 percent reduction in reproduction or growth). A non-quintal

effect is an all or nothing response (e.g., life vs. death, or motile vs. immotile). The LOEC

is the lowest concentration in which a particular effect (e.g., reduced growth, reproduction

or survival) is exhibited at a statistically significant level. The NOEC is the highest

concentration in which no statistically significant effects are observed.

The IC25 is determined by statistical point estimation techniques and may be accompanied

by confidence limits. The NOEC and LOEC are determined by statistical hypothesis testing

techniques and are dependent upon the dilution factor used in a toxicity test. Statistical

confidence limits may not be placed about the NOEC or LOEC.

B. Test Organisms.

The test organisms used for chronic toxicity testing will be Ceriodaphnia

dubia and

Pimephales promelas (fathead minnow).

15

C. Length of Chronic Toxicity Tests.

1. Fathead Minnows:

The length of the fathead minnow chronic toxicity test is 7 days.

2. Ceriodaphnia dubia:

The nominal length of the Ceriodaphnia

dubia chronic toxicity test is 7 days. The end

point of the test involves the production of 3 broods by the control organisms. The

test may end after 6 days if 60 percent or more of the controls have had 3 broods and

have produced an average of 15 young per surviving female, as a minimum. The test

may last as long as 8 days if 3 broods have not been produced by the controls. This

could occur if temperatures were to drop, etc.

D. Type of Test.

Chronic toxicity tests will be static renewal tests. Test solutions should be renewed every

24 hours, using one of three sample sets collected throughout the duration of the test. The

first sample should be used to initiate the test on “Day 0" (start of test) and to renew the

solutions at “Day 1" (24 hours). The second sample set should be collected to renew the

test solutions at “Day 2" (48 hours and “Day 3" (72 hours). The third sample should be

collected to renew test solutions at “Day 4" (96 hours), “Day 5" (120 hours) and “Day 6"

(144 hours). Care should be taken not to exceed the allowable holding time for each

sample (see Section 3.E). Ohio EPA prefers the above sample collection arrangement;

however, if plant operation, scheduling difficulties, or other problems arise, an alternative

sample sequence may be used. If an alternative sequence is used, it should be noted on the

test report.

All chronic toxicity tests are definitive tests, designed to quantify the amount of chronic

toxicity. Therefore, a series of dilutions of the effluent shall be tested using a minimum of

five effluent concentrations for each chronic test. The typical dilution series consists of

solutions of 100, 50, 25, 12.5 and 6.25 percent by volume effluent.

There may be instances when it will be advantageous to use a different dilution series.

Selection of the appropriate dilution series is the responsibility of the permittee. Ohio EPA

staff will be available for consultation on the issue of appropriate dilution series. However,

the permittee, in conjunction with the testing laboratory, may exercise professional

judgement on the selection of appropriate dilution series. When selecting a dilution series,

the following two issues should be considered:

1. Allowable Effluent Toxicity:

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AET is the permissible amount of toxicity for a particular discharge. This value is

determined by factoring the available dilution in the receiving stream with the water

quality criteria for toxicity as well as the effects of any interactive discharges. Should

permit limitations for toxicity be established for a permittee, the AET is the amount

of toxicity that would be allowed. Methods for calculating the AET are contained in

OAC 3745-02-09, as mentioned in Section 2.D.2.a. When selecting a dilution series,

the relationship of that series to the AET should be considered. The series selected

should be one which will yield data to determine if the AET has/has not been

exceeded, yet still identifies the NOEC, LOEC, and IC25.

2. Toxicity in the Effluent:

There may be instances when it is necessary to alter the dilution series in order to

better determine the NOEC, LOEC and IC25. This would be the case in an effluent

that exhibits moderate chronic toxicity. In order to better define the NOEC, LOEC

and IC25, the dilution series has to be shifted to a higher set of dilutions. Also, if

effluent toxicity is consistently exhibited within a certain range, it may be advisable

to adjust the dilution series to focus in that area to better define the NOEC, LOEC

and IC25.

If the chronic toxicity shown by the effluent falls into a large gap in the dilution series

(e.g., between 100 and 50 percent effluent), it may be difficult to discern whether or

not the AET is actually being exceeded. For example, assume a discharge to a stream

has an AET of 1.3 Chronic Toxic Unit (TU

c

) and the dilution series used for testing is

100, 50, 25, 12.5 and 6.25 percent effluent by volume. If the NOEC equals 50

percent and the LOEC equals 100 percent, the TU

c

value equals 1.4 (see Section

3.H.1 for the definition of TU

c

). However, one would be unable to distinguish if the

AET of 1.3 TU

c

was really exceeded. With a dilution series of 100, 75, 50, 25 and

12.5 percent by volume effluent, one would be able to tell if the AET was actually

exceeded. If the LOEC was 100 percent and the NOEC was 75 percent, the AET

would not have been exceeded. However, if the LOEC was 75 percent and the

NOEC was 50 percent, the AET would have been exceeded.

Control and ambient water samples are tested full strength (i.e., no dilutions).

E. Sample Collection.

Effluent samples used to initiate and renew the test solutions in a chronic toxicity test

should be collected as 24-hour composite samples. If the effluent is chlorinated for

disinfection purposes, the effluent sample should be collected at a point prior to

chlorination. The protocols in Section 1.A.1 should be consulted for the handling of a

chlorinated sample if it is impossible to obtain a sample prior to disinfection. However, if

17

dechlorination is an integral part of the disinfection system at the facility, the sample

should be collected at the final outfall.

Unless specifically modified by the NPDES permit, a chronic toxicity test of an effluent

requires that an upstream control sample be collected as well as near-field and far-field

downstream samples during each effluent sampling event. The upstream control sample is

to be collected as a grab sample upstream from the zone of effluent and receiving water

interaction. Care should be taken to assure that any upstream backflow of the effluent is

taken into account when selecting the upstream sampling location.

The near-field downstream sample is to be collected as a grab sample from within the

effluent plume in the immediate vicinity of the outfall. The near-field sample should be

collected in the middle of the effluent plume at a distance of 5 times the water depth at the

point of discharge, down current from the outfall. When water depth at the point of

discharge exceeds 4 feet, the near-field sample should be collected 20 feet (6 meters) down

current from the outfall.

The far-field downstream sample is to be collected as a grab sample within the effluent

plume at a point which represents fairly complete mixing of the effluent and the receiving

water. The available volume of dilution and the mixing characteristics of the receiving

stream influence the selection of an appropriate far-field sampling point. Additional details

are given below:

1. Rapid and Complete Mixing.

In situations where mixing is rapid and complete, the far-field sample should be

collected mid-stream, at a distance of five times the stream width at the point of

discharge, down current from the outfall. Ohio EPA anticipates that rapid and

complete mixing conditions should occur when receiving stream dilution to discharge

ratios are 10:1 or less, and the stream exhibits good pool, run, riffle development. The

presence of rapid and complete mixing should be verified and documented at least

once during the sampling program. If rapid and complete mixing cannot be

documented, sampling should be conducted as if mixing is not rapid and complete.

2. Mixing is not Rapid and Complete

In situations where mixing is not rapid and complete, the effluent plume should be

definitively located during each sampling event. The far-field sample should be

collected mid-plume down current from the outfall at a point five times the stream

width at the point of discharge. When the stream width at the point of discharge

exceeds 500 feet, the far-field sample should be collected mid-plume 2,500 feet (750

meters) down current from the outfall at a maximum. Ohio EPA anticipates that slow

mixing effluents or shore-hugging plumes that persist for considerable distances may

occur when receiving water dilution to discharge ratios are 10:1 or greater, or the

discharge is to a receiving stream that tends to remain in a pooled condition. In these

18

situations, it is imperative that the effluent plume be located and documented for each

sampling event, and that the sample be taken at mid-plume.

The location of the near-field and far-field sampling locations with respect to the effluent

plume must be determined and documented at the time of sampling using temperature

measurements, conductivity measurements, visual observation, a dye study, or other

detailed techniques for delineating the effluent plume. Upon prior approval from Ohio

EPA, an alternative far-field sampling location can be determined from a detailed mixing

zone study.

First use of samples collected for toxicity testing should occur within 36 hours of

completion of sampling.

F. Quality Assurance.

1. Requirements for the Repeating of a Test:

A repeat of a 7-day, short-term chronic toxicity test is mandatory when a combination

of mortality and adverse effects in both laboratory and receiving water controls

exceeds 20 percent for a particular species. A repeat of the test is not necessary if

there is 20 percent or less affected in one of the two control waters.

2. Organism Survival:

The long-term ability of the test organisms to survive can be monitored in a chronic

toxicity test by evaluation of the growth and reproductive success of the control

organisms. Failure of the control organisms to exhibit the following indicators

invalidates the test results and, therefore, a repeat of the test if required.

a. Ceriodaphnia dubia

1. Pretest Culture - Records shall be maintained of the adult Ceriodaphnia

dubia females from which neonates used in the chronic test are to be

obtained. The adult Ceriodaphnia

dubia should be cultured in individual

containers, fed daily, and container solution renewed at least 3 times per

week. If the following conditions are met in the 7-day period prior to

testing, the neonates should be suitable for use:

o

Adult mortality may not exceed 20 percent.

o

A minimum mean number of 20 young per surviving adult in 3 broods

are produced.

19

o

Neonates used in the test are obtained from broods of 8 or more.

2. Controls During Testing - Control organisms shall demonstrate acceptable

survival (see Section 3.F.1). In addition, the controls shall produce an

average of 2.5 broods per test organism with mean production of at least

15 young per surviving animal in the test.

b. Fathead Minnows

Control organisms shall demonstrate acceptable survival (see Section 3.F.1). In

addition, if the larval fish are between 24 and 48 hours old at test initiation, the

controls shall show an average of at least a 3-fold weight increase. If the

fathead minnow larvae are less than 24 hours old at test initiation, the average

dry weight of the control organisms at the end of the test shall equal or exceed

0.250 mg.

3. Dilution Water Substitution:

If a combination of mortality and adverse effects in the upstream control sample

exceeds 20 percent for a particular test organism during a test, or the conditions

specified in Section 3.F.2.a or 3.F.2.b are not fulfilled, an alterative dilution and

control water should be identified and used for subsequent tests. Acceptable

alternative dilution waters may be similar natural waters, rearing unit water,

reconstitute water, or dilute mineral water. The upstream receiving water sample

should still be collected and tested, but the receiving water should not be used as

diluent. Failure to change the dilution water in subsequent tests may result in

invalidation of testing results and a requirement to repeat the test.

4. Number of Organisms:

a. Ceriodaphnia

dubia - 10 organisms is the minimum number that should be used

in each solution of a chronic toxicity test. These organisms should be set up in

10 replicate test chambers for each solution tested (i.e., one per chamber).

b. Fathead Minnows - A minimum of 40 organisms should be used in each

solution of a fathead minnow chronic toxicity test. These organisms should be

set up in a minimum of 3 replicate test chambers for each solution tested.

However, Ohio EPA recommends the use of 4 replicate chambers which results

in a more robust data base.

G. Chemical Analysis.

20

A sufficient volume of effluent shall be collected during each of the 3 composite sampling

periods to allow aliquots to be used in chronic toxicity tests and for chemical analysis. If

no acute effects are seen during the use of the first 2 composite samples for test initiation

and renewal, then only the third aliquot needs to be analyzed for chemical parameters. If

acute effects are documented during the initial or middle stages of the chronic test, then the

aliquot corresponding to the solution causing the acute toxicity shall be analyzed for

chemical parameters. In determining whether an acute effect has occurred in a sample,

Ohio EPA recommends that if mortality and other adverse effects exceed 20 percent, the

testing laboratory consider the issue of acute effects. Ohio EPA feels that this threshold

should be used in conjunction with the professional judgement of the testing laboratory to

determine if an acute effect is being observed.

Bioassay effluent sampling may be coordinated with other permit sampling requirements as

appropriate to avoid duplication. The analyses detailed in the currently effective Part I,

Effluent Limitations and Monitoring Requirements table(s) in the NPDES permit should be

conducted for the effluent sample. In addition, alkalinity and hardness (as CaCO

3

) should

be measured. Chemical analysis must comply with Ohio EPA accepted procedures.

H. Reporting Requirements.

1. Reporting Toxicity Testing Results on Monthly Operating Reports:

NPDES permits require that results of testing be reported on a form acceptable to

Ohio EPA. The results of toxicity tests required under Part I Permit Requirements

shall be reported on EPA Form 4500 (see Attachment 1).

Results for final effluent shall be expressed as toxic units. A chronic toxic unit (TU

c

)

is defined as:

Tu

c

= 100

IC25

For Ceriodaphnia

tests, Tu

c

should also be calculated as:

Tu

c

= 100

square root of (NOEC x LOEC)

Chronic toxic unit values for Ceriodaphnia tests must be calculated based upon the

NOEC x LOEC square root using the most sensitive end point (e.g.,

growth/reproduction or survival) and should be reported. Toxicity test results should

be recorded on the day in which the first sampling event is completed

(i.e., the day

the first composite sample is picked up). If the NOEC equals 100 percent effluent,

the toxicity test result should be reported on Form 4500 as “AA” (below detectable).

21

Form 4500 must be received by Ohio EPA Central Office by the 15th day of the

month following the month in which the toxicity test samples were collected. It may

be necessary to obtain or provide the toxic unit results prior to receipt of the full

laboratory report in order to fulfill this requirement.