1. Whole Effluent Toxicity Basics
Purpose of the training course is to provide an overview of whole effluent toxicity test methods and implementation
Provide a better understanding of scientific underpinnings of WET
Describe uses and limitations,
implementations requirements
describe methods including a demonstration of a tox test
purpose of a Toxicity Reduction Evaluation
Betty Jane Boros-Russo
NJDEP, Office of Quality Assurance
Christopher J. Nally
American Aquatic Testing

2. History
16th century - scientists began testing the lethality of chemical compounds on animals prior to their use on humans for therapeutic purposes
1930’s - some of the first uses of aquatic organisms for testing to determine the causes of observed fish kills
some of the first methods for conducting toxicity tests were published
Most earlier work was with single chemicals and mostly freshwater
June, 1945, Hart, Doudoroff and Greenbanck published “The evaluation of the toxicity of industrial wastes, chemicals and other substances to fresh-water fish. Included a detailed method.
By the late 1960’s, people began noting differences between groups of organisms living in streams based on the absence or presence of wastewater inputs. CA and MI were beginning using it in reg program.
FWPCA of it is the national policy that the discharge of toxic pollutants in toxic amounts be prohibited. Maintained in 1977 and 1987.
1975 EPA published its “Methods for Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians.”
Federal Water Pollution control Act Amendments (Clean Water Act) section 101(a)3 in 1977 stated that it is the national goal that the discharge of toxic pollutants in toxic amounts be prohibited.
This began the use of WET tests for the control of the discharge of toxics into the environment in the 70’s and 80’s First EPA acute manual
1984 and 85 EPA strengthened and outlined its policy for the technical approach for assessing and controlling the discharge of toxic substances TSD published

3. Use of Toxicity Testing in Water Quality Based Toxics Control
To characterize and measure the aggregate toxicity of an effluent or ambient waters
To measure compliance with whole effluent toxicity limits
As an investigative tool and to measure progress in a toxicity reduction program
As an ambient instream measure of toxicity to identify pollution sources
WET is used as part of the water quality based approach to toxics control
It measures the response of exposed aquatic organisms
Used in all these ways / purposes in NJ
Used in ambient investigations in Delaware for ultimately development of WQBELs.
Statements of policy are found in the Surface Water Quality Standards at N.J.A.C 7:9B-1.5(a)3 and the table at 1.14(c) 12

4. NJ WET Program History
Early 1980’s - Acute monitoring and limits used on a routine basis
Began use of chronic monitoring and chronic limits
Group permit challenge on chronic WET
Settlement and initial chronic WET program revisions
Final program revisions adopted
Acute only to start since chronic short term tests weren’t available. Many states started with acute and chronic together later.
In late 1992, adjudicatory hearing requests by a group of municipal dischargers on chronic limits and related TRE requirements
1996 Agreed to disagree on the science. Agreed that changes in program implementation were necessary.
Prior to 1997 several of limit calculation procedures and program implementation requirements were out of date and inconsistent with USEPA guidance and policy.
That adjudication forced NJDEP to reevaluate and update its rules governing WET.Limit expression and calculation, TRE requirements. Program requirements became more specific, increased oversight of lab activity.
Group permit modification of 100 permits, 11 removed limits.

5. Test Species

6. Species Selection
Sensitive species which are easily cultured and readily available year round
Must provide consistent and reproducible response
Also encourage ecologically, commercially and or recreationally important
No one species is always the most sensitive
Species used is dependent upon salinity of receiving water and the state standards
Ecologically cerio mysid and kelp
Commercially abalone
Recreationally trout
For NJ - whether or not to use a freshwater or saltwater test species is dependent upon the salinity of the receiving water that the discharge flows to.
If the discharge is to a storm sewer, you determine the salinity of the receiving water that the storm sewer goes to.
N.J.A.C. 7:9B-1.5(f) specifically statest that “the objective of the Department is to use test species for toxicity testing bioassays that are representative of the more sensitive aqutic biota from the different trophic levels in question. Test species need not be indegenous to , nor occur in the wtaters inqsuetion.
Freshwater means all nontidal and tidal waters generally having a salinity, due to natural sources, of less than or equal to 3.5 ppt at mean high tide. Saline is grater than 3.5 ppt at mean high tide.

7. New Jersey Freshwater Acute Test Species
Invertebrates: (Daphnids)
Ceriodaphnia dubia
Daphnia magna
Daphnia pulex
Fish
Pimephales promelas Fathead Minnow
Oncorhynchus mykiss Rainbow Trout
Salvelinus fontinalis Brook Trout
Daphnids have only been used for acute testing in recent years.
Trout - though they have been in the regulations governing acute testing since the program began, have never been used in NJ.

8. Ceriodaphnia dubia Female approximately 2 mm
The freshwater test organism used both in NJ and most widely across the nation, mostly for chronic testing.
Closely related to Daphnia, but a smaller and have a shorter generation time.
Inhabits lakes ponds and marshes throughout most of the world
Population consist almost entirely of females, the males appear in fall. Production of males appears to be induced primarily by low water temp, high population densities and/or a decrease in the available food.
Cerio’s reproduce only by cyclic parthenogenesis in which the males contribute to the genetic makeup of the young during the sexual state of reproduction.
Filter feeders.
Clutch of 4-10 eggs is released into the brood chamber where they hatch and are released, when the female molts, grows and a new clutch of eggs is released into the brood chamber.
Photo compliments of Marinco Bioassay Laboratory

9. Pimephales promelas Photo by Karen McCabe from Animal Soup
Adults are small fish typically 43 mm to 102 mm, and aceraging about 50 mm, in total length.
Widely distributed in North America. In muddy brooks, streams creeks, ponds and small lakes, is uncommon or absent in streams of moderate and high gradients and in most of the larger and deeper impoundments, and is tolerant of high temperature and turbidity, and low oxygen concentrations.
bait fish
breeding males develop a conspicuous, narrow, elongated, gray, fleshy pad of spongy tubercles on the back, anterior to the dorsal fin, and two or three rows of strong nuptial tubercles across the snout.
Photo by Karen McCabe from Animal Soup

10. New Jersey Saline Acute Test Species
Invertebrates:
Mysidopsis bahia Opossum Shrimp
(Americamysis bahia)
Fish
Cyprinodon variegatus Sheepshead Minnow
Menidia beryllina Inland Silversides
Menidia peninsulae Tidewater Silversides
Menidia menidia Atlantic Silversides
Of the fish, the sheepshead minnow is the most widely used but in general there are very few saltwater fish used for routine monitoring.

11. Mysidopsis bahia Female approximately 6 mm in length
Most frequently used acute and chronic saltwater test species.
Shrimplike crustaceans (Americamysis bahia)
Occurs in salinities primarily above 15 ppt found in greatest abundance in salinities 30ppt. Eggs develop for about 20 days in the marsupium before the yound are released as jubeniles; broods are released at night during molting.
Very cannibalistic
adults 4.4mm to 9.4 mm. Females usually larger than the male. Somewhat transparent but tinted, yellow, brown or black.
Female approximately 6 mm in length
Photo compliments of Marinco Bioassay Laboratory

12. New Jersey Freshwater Chronic Test Species
Invertebrates:
Ceriodaphnia dubia
Fish
Pimephales promelas Fathead Minnow
Algae
Selenastrum capricornutum
Though algae are contained in the program, they are not used.

13. New Jersey Saline Chronic Test Species
Invertebrates:
Mysidopsis bahia Opossum Shrimp
Fish
Cyprinodon variegatus Sheepshead Minnow
Menidia beryllina Inland Silversides
Menidia peninsulae Tidewater Silversides
Menidia menidia Atlantic Silversides
Other
Arbacia punctulata Sea Urchin
Champia parvula Red Macroalgae
Arbacia and Champia are not used in the program.

14. Test Methods

15. Rules for Conducting Toxicity Tests
40 CFR Table 1A
Effective November 15, 1995
Amended November 19, 2002 and effective December 19, 2002
Methods must be followed as they are written
First acute manual was published in 1975.
USEPA added new biolgical testing methods to the list of approved and standardized analytical method for testing wastewater pollutants. This information was published in the Federal Register as a final amendment to 40 CFR 136 and became effective November 15, 1995.
Prior to this none were in EPA regulation though the documents were published.
Standardized 17 procedures, 7 acute tests 4 freshwater and 6 marine chronic.
These methods do not apply to marine waters of the Pacific Ocean.

16. Incorporate by Reference
Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms. 5th Edition, USEPA, Office of Water, October 2002, EPA 821-R
Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. 4th Edition, USEPA, Office of Water, October 2002, October 2002, EPA 821-R
Short-term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms. 3rd Edition. USEPA, Office of Water, October 2002, EPA 821-R

17. USEPA Methods Documents
Health and safety
Quality assurance
Facilities, equipment and supplies
Test organisms and culture methods
Dilution water
Each method document covers at a minimum a section on each topic,
Two chronic methods documents are set up the same way though the acute is somewhat different
Methods include both requirements and recommendations. The intent of the manuals are to use for a wide range of uses. Requirements where necessary for standardization but much of some sections is left to the implementing authority.

18. USEPA Methods Documents (cont.)
Effluent sampling and handling
Endpoints and data analysis
Individual test methods
Report preparation and test review

19. Test Types Acute and Short-term Chronic Tests Test Species dependent
Static non-renewal
Static renewal
Flow through
Test Species dependent
Use dependent
Static non-renewal tests: The test organisms are exposed to the same test solution for the duration of the test. Pros: simple, minimum physical disturbance to organisms. Cons: toxicants may volatilize or degrade during testing.
Static renewal tests: The test organisms are exposed to a fresh test solution of the same concentration of sample every 24-hrs or other prescribed interval by transferring the test organism from one test chamber to another or by replacing all or a portion of solution in the test chambers. Adv.: addresses toxicant degradation/volatilization issue. Disadv: more labor intensive, more physical disturbance to organisms.
Flow through tests: sample is pumped continuously from the sampling point directly to the dilutor system or a grab or composite samples are collected periodically and then placed in a tank to the dilutor system. Adv.: produces exposure more like that of stream organisms. Disadv Significantly more labor intensive and expensive.

20. Test Design 5 Concentrations + Control Replicates
Serial dilution’s of effluent and “control water” (also termed “dilution water”)
Dilution series of 0.5 or greater
Single concentration test
Replicates
Randomization (organisms/chambers)
EPA recommends multi-concentration test with a minimum of 5 test concentrations plus a control.
Dilution factor a minimum of Attempt to bracket any critical concentration.
Single concentration test = pass/fail test not generally recommended.
Replicates 2- 10
Randomization of test chambers, organisms, throughout the test

21. Perspective is Everything

22. Test Conditions and Acceptability Criteria

23. Acute Toxicity Tests Test Procedures Advantages Disadvantages
96 hours or less (species specific)
Mortality is the measured endpoint
For daphnia mortality determined by immobilization
Advantages
less expensive and time consuming than chronic
endpoint is easy to quantify
Disadvantages
indicates only lethal concentrations
only the effects of fast acting chemicals are exhibited
Short Term relative to life cycle.
Acute means a stimulus severe enough to rapidly induce an effect: in toxicity tests, an effect observed in 96 hours or less is typically considered acute. An acute effect is not always measured in terms of lethality.
Acute refers to the exposure not the effect.
Acute tests are conducted on effluents and receiving waters.

24. Acute Test Acceptability Criteria
Minimum control survival at least 90%
Temperature 20 +/- 1o C
Maximum test organism age at start:
14 days for fish
5 days for Mysid shrimp
24 hours for daphnids
Test acceptability criteria determine the validity and acceptability of the test based on the control typically survival,

25. Short-term Chronic Toxicity Tests
Test Procedures
typically 4-10 days
Mortality, growth, fecundity, reproduction
Advantages
more sensitive than acute, assess parameters other than lethality
may better reflect real world
Limitations
more costly and time intensive than acute
more sensitive to low level contamination
Chronic means a stimulus that lingers or continues for a relatively long period of time, often one-tenth of the life span or more. Chronic should be considered a relative term depending on the life span of an organism. The measurement of a chronic effect can be reduced growth, reduced reproduction, etc.., in addition to lethality.
Chronic tests are conducted on effluents and receiving waters.

26. Chronic Test Acceptability Criteria
Minimum control survival 80%
Minimum control dry weight (average):
0.25 mg for fish
0.20 mg for Mysid shrimp
Minimum of 15 young (average) for control C. dubia
Temperature 25 +/- 1o C
Maximum test organism age at start:
48 hours for fish
7 days for Mysid shrimp
24 hours for daphnids
Test acceptability criteria determine the validity and acceptability of the test based on control survival and weight gain.
Example:
The term short-term is used in the WET program
> or = 80& survival
minimum 3 of young / surivivng control female
> or = 0.25 mg wt.

27. Method Specific Test Conditions
Test type and duration
Temperature, light, DO, salinity
Chamber size and volume
Species selection, age and feeding
The test method specifies the minimum number of test organism and the number of replicates required for each test concentration, the range of salinity tolerance of the orgnaism and the test temperature.
The test temperature is specified for each methods and is typically within a narrow ranges. 25+/-1 degree C. Criticalfor chronic can significantly affect growth and reproduction with organisms. Shock due to large increase in temperature.
Light quality intensity and photoperiod are specified for each method.
Do. requirements are specified for each method.DO concentration in the effluent samples should be near saturation prior to used. Aeration may be necessary to bring the DO into equilibrium with air, minimize
DO demand and stabilize the pH. Aeration can reduce the apparent toxicity of the test solutions by stripping them of highly volatile toxic substances, or increase the toxicity by altering the pH. However, the he
DO in the test solutions must not be allowed to fall below 4.0 mg/L. If aeration is necessary the rate is specified in the test and must be conducted equally across all test chambers.

28. Method Specific Test Conditions (cont.)
Dilution water
Dilution series
Sampling
Test acceptability criteria
Test measurements

29. Test Measurements
Dissolved oxygen cannot fall below 4 mg/l (initial and final)
pH (initial and final)
conductivity
total residual chlorine
total hardness and alkalinity
salinity
temperature
Measurements are performed daily to ensure that the test is meeting the requirements of the methods. Some of these can influence test results or mask toxicants.
Low DO caused by high levels of BOD can cause adverse effects to test organisms and mask the presence of chemical toxicants.
pH can modify the toxicity of some compounds. For instance, ammonia is more toxic at a higher pH.
Conductivity can be a general indicator of toxicity associated with total dissolved solids.
TRC is very toxic and can mask the presence of other toxicants.
Hardness of the water can influence the expression of certain compounds. For instance, the bioavailability and thus the toxicity of copper decreases as hardness increases.
For SW tests, the appropriate salinity level must be maintained for the specific marine organisms used in the test.
T is critical in several ways. Test organisms can suffer shock by sudden changes in T. During chronic tests, T can significantly influence growth and reproduction of the test organisms.

30. Selection of Dilution Water
May be either a standard laboratory water or the receiving water
Choice of water is dependent on the objectives of the test
Absolute toxicity use standard water
Estimate of toxicity in uncontaminated receiving water, use receiving water
Contaminated receiving water, use laboratory water
Age - EPA policy is to protect the most sensitive life stages of fish and aquatic life. EPA recognizes that the sensitivity of organisms to toxicants generally decreases significantly with age beyond the early life stage and therefore reduced ages of test organisms in the latest revision from 90 days to 14 days. Fatheads and inland silversides.
Use of receiving water may require a second set of controls with culture water.
Acclimation of organisms to salinity
forty fathoms
Brine from saltwater
Spring water
DI water
Use of receiving water in a test for controls generally requires the use of a second set of controls using standard laboratory water.
Effluents are typically freshwater and are not appropriate for the use of saline test organisms. This necessitates the adjustment of the effluent with dry artificial sea salts. Can result in artifactual toxicity. Increase pH least to increase in ammonia.

31. Data and Endpoints

32. Acute Test Endpoints
LC50 - Concentration of effluent that is lethal to 50 percent of the exposed organisms at a specific time of observation (e.g. 96 hr LC50), (expressed as % effluent)
NOAEC - No Observed Adverse Effect Concentration
Lowest concentration at which survival is not significantly different from the control
always set equal to 100% effluent
EC - Effect Concentration
LC = Lethal Concentration - point estimate of the toxicant concentration that is lethal to 50% of the organisms during a specified test period. when mortality is measured
EC = Effect Concentration - point estimate of toxicant concentration that would cause an observable adverse effect such as death orimmobilization or serious incapacitation in a given percentage of test organisms. usually for quantal data such as or fertilization

33. Test Data
Typical dose response where mortality increases as the concentration of effluent in the mixture increases.
LC50 would be somewhere between 25% effluent and 50% effluent.
calculated point estimate or a
Pass fail test where a concentration, usually that which is considered a critical concentration of effluent in the recieving water or
ambient toxicity test measured against a control
Examples of Pass/Fail Acute test
Instream waste concentration equals 75%
statistical evaluation using a student-t test compares mortality rates of ambient or IWC sample against a control
Is there a “significant statistical difference between the two results”
6.25 % Effluent
12.5 % Effluent
25.0% Effluent
50.0% Effluent
100.0% Effluent
Control
0% Mortality
0% mortality
20 % Mortality
40% Mortality
80% Mortality
100% Mortality

34. Chronic Test Endpoints
IC25 - Inhibition Concentration - Concentration of effluent which has an inhibitory effect on 25% of the test organisms for the monitored effect, as compared to the control (expressed as % effluent).
NOEC - No Observable Effect Concentration - Highest concentration of effluent tested which shows no statistically significant effect on the organisms as compared to the control (expressed as % effluent).
IC = Inhibition Concentration - % reduction is nonquantal continuous measurements such a length, weight gain, reproduction.
Advantages include CV an SD
Results are often higher for the IC25 than they are for an NOEC
LOEC lowest Observed Effect Concentration the lowest concentration of an effluent or a toxicant that results in observable adverse effects in teh aquatic test organisms.
NOEC and LOEC are hypotheseis testing techniques while the ICp values are point estimates.
NOEC may represent a different amount of effect from test to tests.
NOEC is a lower concentration level than the LOEC
NOEC do not allow calculateion of coefficients of variation or confidence intervals.
ICp you can calculate CVs and CIs.
Always estimating the same effect.
Need to specify the biological effect (the value of p)

35. Chronic Test Data % % Average Effluent Mortality Dry weight % w/Eggs
NOEC % % %
IC % % %
Exclusion of data from animals that do not survive to the end of the test would bias the results in favor of animals that do.

36. Toxicity Values
LC50, IC25, NOAEC: As a limit these values will INCREASE as the limit becomes more stringent
These are minimum limits
LC50, IC25: When evaluating data, exhibit more toxicity as the values decrease
Toxic Units: Maximum limits
As values increase as limits, they become less stringent

37. Toxic Units (TU’s) Reciprocal of the fractional LC50, NOEC, IC25 value
Calculated by dividing the value into 100
TUa = 100/LC50
TUc = 100/IC25
Examples of Toxic Units
Acute (TUa)
Assuming LC50=28%
TUa = 100/LC50
100/28 = 3.6
Assuming LC50=10%
100/10 = 10
Chronic (TUc)
Assuming NOEC=50%
TUc = 100/NOEC
100/50 = 2
Assuming IC25 = 30%
TUc = 100/IC25
100/30 = 3.3

38. Standard Reference Toxicant Program

39. Standard Reference Toxicants (SRT’s)
Purpose
Frequency
Acceptability Criteria
Control Charts
The most important part of the QC program
Mandated for chronic only
SRT program serves to track laboratory’s ability to consistently conduct the test over time, as well as to assess the health of the test organisms.
Potassium chloride is used by most laboratories.
Results of ongoing testing must fall within the control limits established by the data. The control limits are +/- two standard deviations around the mean.
Results outside two standard deviations imply that something has changed.
What is a SRT tests
Why do we run them
What are the requirements
How Frequently are they run
What is a control chart
What should you look for Assure that the labs can consistently perform the tests.

40. Control Charts

41. Sample Collection

42. Subchapter 9 N.J.A.C. 7:18 Subchapter 9 Sample Requirements
Addresses collection, handling and preservation of environmental samples
Section 9.5 – Requirements for acute toxicity testing samples

43. Grab vs. Composite Grab samples offer “snap shot” of effluent
Composite samples offer “average view” of effluent
NJDEP requires sampling based on discharge type
Continuous discharge – 24 hour composite sample
Intermittent discharge – grab or composite each day that is representative of discharge
Type of sample and frequency of collection is dependent upon the use of the data and the discharge the test is intended to represent.
Samples are not to be used after they have been held for 72 hours.
Samples are to be chilled during or immediately upon collection to 4oC.

44. Frequency and Holding
Daily for acute toxicity testing (single comp. for daphnids)
Every 48 hours for chronic testing
Minimum # of samples for 24 hour composite - 48 (every 30 minutes)
Holding times
24 hours to first use for acute and chronic testing
72 hours to use three times, chronic testing only

45. Effluent Sampling Containers
Constructed of non-toxic materials:
Glass – borosilicate, tempered or soda lime
304 or 316 stainless steel
Medical or food grade silicone
Perfluorocarbons – Teflon, etc.
Plastics – polyethylene, polypropylene, polycarbonate,polystyrene
Containers rinsed with sample, used once and disposed of, or cleaned.
Avoid splitting samples across weeks or months.
Sampling days are an issue.

46. Sampling Location
NJPDES sample location must be used for toxicity test sampling
This is generally the same sampling location required for all other parameters
Prechlorination sampling may be required
Post dechlorination sampling may be required
Location should always be specified in the permit
Holding times for acute testing 24 hours to first use;
Holding times for chronic testing 24 to first use, 3 uses in 72 hours
Very important to use NJPDES sample site to make data comparable to chemistries done

47. Preservation
For toxicity testing only temperature preservation permitted
Refrigeration during sampling optional
Refrigeration or icing immediately upon collection required
Sampling can be refrigerated, but not mandatory for NJDEP, sample must be iced immediately upon collection.

48. SAMPLING DOCUMENTATION
Chain of Custody
Facility information
Date, time, sample ID, sampler ID, sample location information
Signatures for custody transfer
Signatures are important!!
Avoids confusion
Prevents sampling from occurring when plant not operating normally

49. New Jersey Toxicity Testing Program

50. Whole Effluent Toxicity Approach to Water Quality Based Toxics Control
WET is used as an effluent parameter to measure the aggregate toxic effect of the discharge of toxic pollutants to surface waters
Goal is to protect aquatic biota and achieve surface water quality standards
Limits are set to be met at the “End of the pipe” to satisfy the “No toxics in toxic amounts” narrative water quality standard
Much of the 70’s in environmental protection was spent evaluating the presence of individual chemicals in wastewater discharges.
By the 1980’s it was widely recognized that a shift in environmental protection was necessary. Protection should be achieved through not only chemical specific limits but also whole effluent toxicity tests. Permittees could be in complianace with chemical specific limits but the effluetn would still be toxic.
Cost effective approach to controlling toxicis.
The test predicts the toxicity of the entire solution the environment taking into account matrix effects and the exposure instream.
1980’s also brought about some of the TRE/TIE testing procedure development
WET is used in discharge monitoring and permitting in combination with the chemical specific program where numeric criteria for various chemicals are used to limit the discharge.

51. The Whole Effluent Approach Capabilities
Toxicity of all effluent constituents are measured and the toxic effect can be regulated with one parameter
Implements the national policy of no toxics in toxic amounts
Chemical interactions are assessed
Unknown toxicants are addressed
Bioavailability of toxic constituents is assessed and the interactions of constituents accounted for
Cant limit everything
Don’t know what everything is.
Where toxicants are additive can provide a better measure
The studies conducted establish results which are predictive of impacts if the appropriate assumptions are met.
Biological criteria
Reflect overall ecological integrity but the problem is that impacts have already occurred therefore they are not predictive, and difficult to interpret impacts.

52. The Whole Effluent Approach Limitations
No direct human health protection
Carcinogenicity, mutagenicity and bioaccumulations are not assessed
No direct treatment
Predictivity of results should be carefully assessed
No persistency on sediment coverage
Incomplete knowledge of a causative toxicant
Not designed to be quantitative predictors of ecosystem response though many studies have demonstrated significant associates between toxicity test results and ecosystem impacts.

53. Program Structure Compliance Testing Program (Enforcement)
Laboratory Certification Program for Acute and Chronic Toxicity
Permit Program
Whole Effluent Toxicity Limits
Toxicity Testing Monitoring Requirements
Toxicity Reduction Evaluations
Compliance Testing Program (Enforcement)
All aspects covered that are recommended by EPA
Lab cert program for acute since early 80s chronic was not until 2001
Prior to that chronic was enforced through permits.
Compliance testing program for toxicity has been in existence since the very early stages of the program, with one of the first industrial discharges to receive a limit, eventually paying penalties for violations of the toxicity limit.
Who each program is run by

54. Laboratory Certification Program
Regulations Governing the Certification of Laboratories and Environmental Measurements (N.J.A.C. 7:18)
Subchapter 7 contains test methods
Subchapter 9 contains the procedures governing sample collection and handling
Formal certification program
National and state programs
Governs many other programs
Both a local and national program NELAP
Lab must apply to the Department
Pass an onsite inspection
Review of SOPs and Quality system

55. WET Certification Components
Personnel qualifications
Laboratory facilities and safety
Equipment and instrumentation
Sample collection, handling and preservation
Test Methodology
General lab practices
Quality control
Reference toxicant data
Records and data reporting
Test acceptability criteria
Comprehensive set of requirements
Personnel minimum educational and experience requirements.
Number of replicates, concentrations and controls.
Types of water used for culturing and testing.
Types of glassware which can be used as well as the cleaning procedures for that glassware.
Sample types (composite or grab), holding times and temperatures.
Minimum number of replicates, types of test organisms, age of organisms.
Maintenance of records, QA manual, SOPs.

56. Acute Toxicity Methods
Methods contained in rule at N.J.A.C. 7:18
5 concentrations + control
replicates
96 hours or less (species specific)
Mortality or immobilization
Receiving water for dilution preferred
Test species
Acute is more detailed in equipment, dilution water and sample collection. However, the test methods themselves are not significantly different.
3.5 ppt
Most common freshwater test species are Ceriodaphnia dubia and Pimephales promelas (fathead minnow).
Most common saline test species are Mysidopsis bahia and Cyprinodon variegatus (sheepshead minnow).
Other test species are used in the program and are based on sensitivity and site characteristics, such as salinity.
Static renewal and flowthrough
Differences from EPA
Problems with dilution water collection

57. Chronic Toxicity Methods
Incorporated by reference in N.J.A.C. 7:18-7.1(a)
Part V includes additional requirements
USEPA Methods (40 CFR 136)
Certified laboratories
Same test species as acute testing
“Part V.”
This document specifies the methods applicable to the NJ WET program, outlines the Standard Reference Toxicant (SRT) Program and defines areas left to the NJDEP by EPA such as effluent sampling, use of dilution water, test acceptability criteria and reporting.
Fecundity endpoint is not required for the mysid test
Sampling again is the main concern - issue
3 samples collected during the test - once every other day
problems

58. Permitting

59. Limit Calculation
N.J.A.C. 7:14A-13 - Effluent Limitations for DSW Permits
USEPA’s Technical Support Document
Acute and Chronic WQBELs
Values of 0.3 and 1.0 used to interpret narrative “no toxics” criteria
Reasonable Potential determinations based on site specific data
Effluent toxicity standard at N.J.A.C. 7:9-5.7(a) is an LC50>50% effluent
Technical Support Document for Water Quality-based Toxics Control (EPA/505/ ) march 19___.
In 7:14A specific section on determining RP, specifically states if max exceeds or max projected exceeds you must impose limit
Get more data if insufficient data exists.
Steady state mass balance - no accounting for background toxicity and generally complete mixing is assumed.
Regulatory Requirements for Development of WQBELS for WET are found in 40 CFR (d)1.
Collect data and determine reasonable potential to exceed Water quality standards using numeric or narrative criteria. Account for existing controls on point and nonpoint sources of pollution, the variability of the pollutant and the sensitivity of the test species.
Effluent and receiving water flow, available mixing
acute and chronic mixing zones, which is more protective
limit adjusted based on data variability
limit is evaluated for RP

60. Who Gets What??
What gets imposed - acute / chronic, limits or monitoring only, is highly dependent on what type of data is available
Generally, if no data exists a limit will not be imposed right away, unless the discharge is the result of a cleanup
Final limits are affected significantly by available dilution and the acute:chronic ratio
Every discharge gets evaluated to some extent for WET- data submitted with application.
Only a single limit is imposed
Same procedure used to calculate other WQBELs
Where only acute data is available, generally the permit will contain additional data collection requirements.
What gets imposed is entirely dependent on what data is available
Limits not imposed without a Reasonable Potential Analysis
Affected significantly by available dilution and acute:chronic ratio (ACR)
Where chronic data is available, adequate data is usually available to calculate and determine reasonable potential. It is in this case that a limit may be imposed.
Short duration batch discharges generally do not receive chronic testing requirements.

61. Limits - The Bottom Line
Variability of data affects the final limit outcome
The more data the better
Ensure input values are appropriate
Variability of toxicity test results will influence permit development
Minimal variability and high quality effluent will lower reasonable potential
Reasonable potential is influenced by dilution, need to accurately assessed and modeled
Collect data under all conditions, wet or dry weather, high or low flow
Don’t stop at the minimum
Experience, culture history and health (ref tox results), visit the lab, references, experience with TIEs,
Be proactive

62. Permit Requirements Limit and testing frequency
Test species and method
Reporting requirements (endpoints)
Repeat testing requirements
Characterization requirements
Split samples
Toxicity Reduction Requirements
Compliance schedule vs. trigger
Interim vs. final limits
3 or 5 years
Limit, monitoring frequency and effective date in Part III, effluent limitations table.
3.5 ppt
For any permit containing a toxicity limit, there is an associated section in Part IV of the permit.
Monitoring frequency is generally based on tables contained in rule at 7:14A-14. Quarterly testing for major facilities an semi-annual for minors. Can be reduced after several years of data has been collected if no reasonable potential can be supported.
Where violations occur of an effective limit, the penalty rules at 7:14-8 require monthly monitoring. For limits not effective TRE requirement would apply.

63. Toxicity Reduction Evaluations (TRE)
Specific TRE language is included at N.J.A.C. 7:14A-13.17(a)
Language to exclude test results not considered representative included at N.J.A.C. 7:14A-13.14(a)2
Permittees responsibility
NJDEP oversight role
Series of stepped requirements
Apply whether limit in effect or not
Compliance schedules of three years are routinely incorporated into permits.
The TRE requirements consist of a series of stepped requirements based on an initial trigger of 2 violations in 6 consecutive tests. The first step (Toxicity Characterization Phase) is a period of more frequent monitoring. The second step (Preliminary Toxicity Investigation) is an evaluation of in plant toxicity controls and assessments. The third step (Comprehensive Toxicity Investigation) is a detailed formalized Toxicity Identification Evaluation.
Intent of the requirement is that the limit has been imposed based on site specific data which has exhibited “reasonable potential to violate water quality standards.” So if the permittee begins monitoring under a new permit, the three year compliance schedule provides adequate time to determine if an intensive TIE is necessary. The permit allows for extension of compliance schedules to five years where permittee is in a “good faith” TRE.

64. Regulatory Issues Recent Developments
Toxicity test precision can be compared to analytical chemical precision.
Analytical chemical precision similar to toxicity test inter-laboratory.
Most commonly used test spec. Ceriodaphnia dubia. Many issues raised regarding culturing, methods and data analysis. Presence of males microscopic examination Test termination, 60% of 3 brood.

65. USEPA Support for WET
EPA National Policy for WQBEL development for Toxic Pollutants
CFR Revised for WQBELs
Technical Support Document for Water Quality-based Toxics Control
WET Control Policy Updated
Incorporation of WET methods in 40 CFR 136
1984 Policy for the Development of Water Quality based Permit Limits for Toxic pollutant addressed the use of biological and chemical methods to assure that toxic whole effluent discharges were regulated consistent with federal and state requirements. Based on sections 101(a) and 308 of the CWA, Who should be reviewed for WET what data to consider how to do reasonable potential monitoring and compliance schedules.
1989 revised federal regs which contained non-specific requirement that NPDES permits contain any more stringent requirements necessary to achieve state WQS. Codified in 40 CFR. The preamble detailed explanation of the legal and policy support for WET testing and limits.
1991 TSD revised with detailed guidance
July 1994 updated policy in (d) governing the development of limits for WET reaffirming their strong commitment to control WET under existing regs.
October 16, CFR updated effective November ‘95.

66. October 26, 1995
40 CFR revised to establish standard protocols for conducting WET tests
Incorporates acute and chronic test method manuals by reference
Supplemental Information Document provides responses to comments raised
Revisions to Part V to reference 40 CFR 136
In November 1995 USEPA issued a final rule standardizing WET testing procedures for use in NPDES monitoring. The rule set uniform WET test procedures for determining the Toxicity of the overall effluent. 17 different procedures, 4 freshwater chronic, 6 marine chronic and 7 acute.
In comments received on the draft rule as well as the response to the final rule, there were complaints of inconsistency of the methods and that the methods were to variable for use.
6 different litigants sued EPA in 5 different courts of appeal. (Western Coalition of Arid States (WESTCAS) and the Edison Electric)

67. NJ WET Program History
Early 1980’s - Acute monitoring and limits used on a routine basis
Began use of chronic monitoring and chronic limits
Group permit challenge on chronic WET
Settlement and initial chronic WET program revisions
Final program revisions adopted
In late 1992, adjudicatory hearing requests by a group of municipal dischargers on chronic limits and related TRE requirements
1996 Agreed to disagree on the science. Agreed that changes in program implementation were necessary.
Prior to 1997 several of limit calculation procedures and program implementation requirements were out of date and inconsistent with USEPA guidance and policy.
That adjudication forced NJDEP to reevaluate and update its rules governing WET.Limit expression and calculation, TRE requirements. Program requirements became more specific, increased oversight of lab activity.
Group permit modification of 100 permits, 11 removed limits.

68. Settlement Agreement Requirements
July 24, 1998
Variability Guidance Document
Method Guidance Document
Interlaboratory Variability Study
Rulemaking actions
Law suit settled by 3 different settlement agreements, each requiring specific EPA actions to complete several products.
It is not a consent decree, which would be independently enforceable
existing test manuals to clarify mandatory items to assure consistency in measurements and clarify discretionary options to optimize successful test completion.
Rulemaking to convert discretionary provisions:
Ceriodaphnia test randomization and blocking by parentage
Development of valid dose concentration response as a re-requisite for a valid test
pH control / shift
Prepare guidance and recommendations regarding adjustments to statistical error rate assumptions, confidence intervals, available dilution waters and permutation of the valid dose-response curve. The agreement require that these actions be announced in January 2000
In JANUARY Revise manuals to incorporate new requirements and recommendations
Current methods provide sufficient direction and flexibility until that time.
Recognize variability arises from effluents, analytical procedure and the analyst
Develop guidance on how to take analytic variability into account for determining the need for and derivation of the WET Limit by April 2000

69. Results 8 of 10 methods had test completion rates >90%
Test completion rate of 82% for Ceriodaphnia
Successful test completion rate of approximately 64% for Selenastrum
7 of 10 test with no false positives
9 of 10 methods had false positives<5%
82% = 8 labs failed 50% or more of tests initiated, 24 labs had no failures
for selenastrum - possible explanations for test failures
Stock culture health - tests initiated on the same day in a laboratory either both passed or both failed
stock culturing technique - labs that culture organisms with EDTA often failed a high percentage of tests without EDTA, labs that cultured organisms without EDTA often failed a high percentage of tests with EDTA
CVs were below 50% for all tests except selenastrum
Lower than average values cited in the method manuals for the same methods at the time of method approval.

70. Conclusions
WET Variability Study results confirmed EPAs conclusions that WET methods provide sufficient precision and can be reliably used in permits
In September 2001, EPA proposed to ratify its previous approval of the methods evaluated in the study
Hope that this study will advance the WET debate from questions of method variability to approaches for improving the implementation of the WET program.

71. Technical Corrections Notice
February 2, 1999 (64 FR 4975)
Incorporated into the WET final rule an errata document
corrects minor errors and omissions
provides clarification
established consistency among the methods manuals and the final rule

72. Variability Guidance Document
July 18, 2000 (65 FR 44528)
Guidance to regulatory authorities, permittees, and testing labs on measurement variability in WET testing
Explains the toxicity test protocol, organisms, chemical and physical conditions, renewals, dilution series, test design, measurements (mortality reproduction) data analysis and test endpoints
Understanding and Accounting for Method Variability in WET Applications Under the NPDES Program.
Quantifies the variability of WET methods and provides guidance for addressing and minimizing variability.
Contains CVs for promulgated methods
Evaluate statistical methods described in the Technical Support Document for determining RP and deriving WET permit conditions.
EPA prepared a document which was then peer reviewed the document 5 independent experts not identified to EPA staff.
Peer reviewed conclusions: substantial agreement with approach detailed comments greatly improved the document.
Rather extensive document.

73. Method Guidance Document
July 28, 2000 (65 FR 46457)
% Minimum Significant Difference
Confidence intervals
Concentration response relationship
Dilution series selection
Dilution water selection
Provides specific technical guidance on issues of WET test conduct and result interpretation
Completed July 28, 2000
%MSD calculated for any test should be less than or equal to a a based on test species should be considered for all test results
CR must be considered in all cases, Not all tests will produce ideal, classical CR curves. Examples of CR curves and guidance on whether to use the data.
The evaluation of CR relationships is not always clear-cut
Look at variability study data, 698 samples 30 were flagged and recommended resetting
Typical 6.25, 12.5, 25, 50, 100 or 0.5 dilution factor not required don’t be afraid to change, consider the effluent limit, data variability, objectives of the test.
Dilution water of consistent quality, meets objectives of the test, organisms perform adequately. Adequate flow year round, support adequate performance of test organisms, consistent quality without contaminants.
Dual controls - how to evaluated the data.
Incorporate guidance into final changes to methods.

74. Laboratory Errors
Errors in the analysis and reporting of WET test results were prevalent.
Errors ranged from single data entry or rounding errors to errors in statistical method selection for use.
Most errors had minor effects on test results.
Lab data submitted for interlaboratory study were reviewed to evaluate the accuracy with whidch the labs routinely analyzed and reported WET test results.
Most errors wer made in the calculation or reporting of results.
Errors ranged from simple data entry or computational errors to misinterpreation of test endpoints or misuse of statstical methods.
Many of these errors were minor and resulted in little or no difference in the reported result.
These results indicate a need for increased lab quality control and personnel training, with an increased emphasis on state and regional lab cert programs, and increased client attention to lab selection and result reporting.

75. Guidance to Testing Laboratories
Maintain QC control charts for IC25 PMSD.
Routinely plot average treatment responses and replicate data to identify anomalies and excessive variability.
Ensure that the upper PMSD is not exceeded.
Use at least four replicates for minnow tests.
Additional topics: lab quality control, standardizing reference toxicants, acceptance limits for ref-tox test results.

76. Guidance to NPDES Permittees
Use one laboratory
Review your laboratory’s control charts
Check test acceptability criteria
Check sample holding times and Chain of custody’s
Obtain at leas 10 data points over >1 year to characterize effluent variability

77. Guidance to Regulators
Review the test reports
Evaluate PMSD as well as TAC
Conduct routine lab audits
Review SRT control charts

78. Proposed Rule Amendments
September 28, 2001 (66 FR 49794)
Specific revisions to the test methods and proposed to ratify its previous approval of the methods
Comment period scheduled to end on November 27, 2001, extended to January 11, 2002

79. Final Rule Issued November 19, 2002 Vol. 67. No. 223, 40 CFR 136
Effective December 19, 2002
Ratified most of the previously adopted methods
Amended the table containing the toxicity methods
All of the methods commonly used in New Jersey were adopted.

80. Ratification of Ten Methods
Methods are repeatable and reproducible
Available and applicable
Representative
Variability study showed high rate of successful completion
Do not often produce false positive results
Exhibit precision comparable to chemical methods approved at 40 CFR 136
methods are repeatable and reproducible (i.e.. Exhibit adequate within-laboratory and between-laboratory precision)
Available and applicable (I.e., adaptable to a wide variety of laboratories and use widely available organisms and supplies
representative (predictive of receiving system impacts.
Variability study showed high rate of successful completion
do not often produce false positive results,
exhibit precision comparable to chemical methods approved at 40 CFR 136

81. Withdrawal of Two Methods
Holmesimysis costata Acute Test
west coast test organism
Champia parvula Reproduction Test
Methods can still be used
EPA was unable to obtain interlaboratory precision data for these methods in the WET Interlaboratory Variability study due to laboratory unavailability.
EPA was unable to contract with a minimum of 6 labs qualified and willing to conduct these test methods within the time frame of the study.
By withdrawing methods from 136, EPA does not reject their use on more limited bases.
Agency has not validated the methods for national use
EPA continues to support the use for applications other than for the determination of compliance with NPDES permit limits, as well for limited, localized, or regional use where the methods have been validated by other entities.
Holmesimysis costata pacific ocean test organism, test organisms not listed in 40 cfr 136.

82. Amendment to 40 CFR 136.3 Table 1A
Clarified mysid test method does not apply to Holmesmysis costata
Added method numbers to acute tests
Modified footnotes and references to cite the updated version of the method manuals
Revise the parameter measured in marine tests to refer to organisms “of the Atlantic Ocean and Gulf of Mexico”

83. Impact of the Adoption Blocking by parentage
Ceriodaphnia test endpoint
pH drift
Dilution series
Dilution water
Pathogen interference
Variability criteria
Minimum number of replicates
Test requirements / recommendations
Reference toxicant testing
Sample collection and holding times
Sampling holding temperature
Biomass
Total residual chlorine
Additional minor corrections
States no one dilution series is required. Recommendations only.
No single dilution water type is required. Synthetic or nature waters may be used as long as they meet the requirements for acceptable dilution water which is one which supports adequate performance of the test organisms with respect to survival growth, reproduction or other responses that may be measures in a test, is of consistent quality and doesn’t contain contaminants that could produce toxicity.
Mandates the use of variability criteria for five chronic methods. Recommends use of point estimation.
TRC only required where expected to be present

84. Ceriodaphnia dubia Chronic Toxicity Test
Mandated use of a very specific procedure of “Blocking by Known Parentage” with at least six neonates
Neonates from a single parent may be used to initiate more than one test
Elimination of use of fourth brood organisms
Blocking requires the use of at least six neonates from each of at least ten separate parents.
If more than 6 neonates from a given parent remain after allocating organisms to a test, those remaining neonates may be discarded, used in future culture organism, or used in another test initiated on the same day (must be 24 hr age requirements.

85. pH Drift Changes between proposal and adoption
Permitted in chronic methods only
Specific procedures to demonstrate need for pH control
Specific procedures for pH control during testing
Clarifies that when the test objective is to determine the absolute toxicity of the effluent, the target pH to maintain in a pH-controlled test is the pH of the sample upon completion of collection.
Clarify that in pH-controlled tests, pH should be maintained within +/-.2 for fw and .3 for SW.
Guidance on interpreting the results in parallel testing.
Clarified that they did not believe that pH drift alone is not a test interference if pH is within the organism’s tolerance range. The Degree of pH drift typically observed in effluent samples should generally only interfere with test results if the sample contains a compound with toxicity that is pH dependent and at a concentration that is near the toxicity threshold. Because EPA did not receive data to suggest otherwise, EPA removed any reference to pH drift interference in the absence of pH dependent toxicants.
No pH control guidance is included in the acute test methods because those methods pH drift may be remedied by more frequent test renewals or the use of follow-through testing.

86. PMSD Percent Minimum Significant Difference
Test Method: Endpoint 10th PMSD 90th PMSD
Fathead Minnow Growth
C. dubia Reproduction
Sheepshead minnow Growth (6.3) (23)
Inland Silverside Growth
Mysid Growth
PMSD values calculated with Dunnett’s test must be between within the range established by the 10th and 90th PMSD values.
PMSD = (MSD x 100) / Control Mean (This value is calculated via the Dunnett’s program)

87. Test Requirements / Recommendations
Modification of tables summarizing test conditions
New section on test review
Mandatory review of concentration response relationship by regulatory authority
Mandatory use of variability criteria for data reported as an NOEC

88. Reference Toxicant Testing
Used for initial and ongoing demonstration of performance and to assess sensitivity and health of test organisms
Monthly or side by side testing
Use of suppliers five most recent tests
Not a “de facto criterion” for test rejection
Labs should evaluate CVs based on national values

89. Sample Collection & Holding
Maintained default maximum of 36 hrs for first use of sample
Clarified these samples may be used for later renewals
Permitting authority may allow continued use of most recent sample
Collection on days one, three and five recommended (not required)

90. Enforcement

91. Serious Violation Limit (% Effluent) > or = 80 and < or = 100
Result Difference
> or = 20
> or = 15
> or = 10
> or = 9
What is deemed a violation of a toxicity limit is defined in N.J.A.C 7: Civil Administrative Penalty for Violation of Whole Effluent Toxicity Limits.
A Serious Violation has a mandatory minimum penalty of $1000 for the first violation and $5000 for each subsequent violation. Penalty maximum can be not be greater than $50,000 for a serious violation. Non serious penalty maximums can not be greater than $40,000.

92. Affirmative Defense
N.J.A.C. 7: violator is entitled to an affirmative defense to liability for a violation occurring as a result of an upset, an anticipated or unanticipated bypass, or a testing or laboratory error.

93. Testing or Laboratory Error
A violator asserting a testing or laboratory error as an affirmative defense shall also have the burden to demonstrate that a violation involving the exceedance of an effluent limit was the result of unanticipated test interference, sample contamination, analytical defects, or procedural deficiencies in sampling or other similar circumstances beyond the violators control.

94. FAQ’s

95. Toxicity where you don’t expect it??
Stormwater
Cooling Water
Filter backwash
High or low TDS
Chlorine

96. Naturally low pH water N.J.A.C. 7:18-9.5(a)2vii.
If the receiving water has a natural pH below 5.0 units, then the dilution water samples shall be adjusted to a pH of 5.0 prior to their use in test organism acclimation and/or toxicity testing.

97. Where can I get toxicity data on various chemicals?
The ECOTOX (ECOTOXicology) database provides single chemical toxicity information for aquatic and terrestrial life. ECOTOX is a useful tool for examining impacts of chemicals on the environment. Peer- reviewed literature is the primary source of information encoded in the database. Pertinent information on the species, chemical, test methods, and results presented by the author(s) are abstracted and entered into the database. Another source of test results is independently compiled data files provided by various United States and International government agencies.

98. Other Issues NMAT to NOAEC How much data is enough? Dilution estimates
When is data too old?
What are other states doing?
Intermittent discharges?
Sampling issues for intermittent discharges.

99. How does increasing the difference in test concentration dilutions affect the prediction of response?
Better resolution around threshold effect concentration
Reducing the distance between effluent dilutions should be encouraged
minimum set of dilutions, i.e. no wider than 0.5 dilutions between concentrations
Test design should maximize test concentrations around the instream waste concentration, in order to minimize the need for interpretation of effects between tested concentrations
Better resolution around threshold effect concentration provide better input to mathematical models to predict point estimations of effect and reduce uncertainty in hypothesis test of effect. Reducing the distance between effluent dilutions should be encouraged. There may be some confusion about EPA’s specification of dilution series in these cases. The methods specify a minimum set of dilutions, i.e. no wider than 0.5 dilutions between concentrations. No limitations on added concentrations within that range exist. Experimental design should account for concentrations of concern and should attempt to maximize resolution in that range. Test design should maximize test concentrations around the effect conc.. Of concerns, I.e the instream waste concentration or limited concentrations of a discharging facility, in order to minimize the need for interpretation of effects between tested concs.

100. My effluent tests indicate there may be a problem but I can see fish in the area of my discharge, is there really a problem?
Observations of organisms in the area of the outfall does not mean that more subtle impacts are not occurring or that the organisms that are present are sensitive enough to represent most organisms instream.
Reduction of a food source
Presence of pollution tolerant organisms
more sensitive organisms are already gone
Sometimes difficult to establish agreement between tox test results and instream conditions.
Limits are based upon available dilution, effluent variability.

101. WET Resources www.epa.gov/waterscience/WET