Whole Effluent Toxicity Basics

1
Whole Effluent Toxicity Basics

• 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
• 1930s - some of the first uses of aquatic
  organisms for testing to determine the causes of
  observed fish kills
• 1945 - some of the first methods for conducting
  toxicity tests were 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

4
NJ WET Program History

• Early 1980s - Acute monitoring and limits used
  on a routine basis
• 1989 - Began use of chronic monitoring and
  chronic limits
• 1993 - Group permit challenge on chronic WET
• 1996 - Settlement and initial chronic WET program
  revisions
• 1997 - Final program revisions adopted

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

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

8
Ceriodaphnia dubia

• Female
• approximately 2 mm

Photo compliments of Marinco Bioassay Laboratory
9
Pimephales promelas
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

11
Mysidopsis bahia
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

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

14
Test Methods
15
Rules for Conducting Toxicity Tests

• 40 CFR 136.3 - Table 1A
• Effective November 15, 1995
• Amended November 19, 2002 and effective December
  19, 2002
• Methods must be followed as they are written

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-02-012
• 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-02-013
• 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-02-014

17
USEPA Methods Documents

• Health and safety
• Quality assurance
• Facilities, equipment and supplies
• Test organisms and culture methods
• Dilution water

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
• Static non-renewal
• Static renewal
• Flow through
• Test Species dependent
• Use dependent

20
Test Design

• 5 Concentrations Control
• Serial dilutions of effluent and control water
  (also termed dilution water)
• Dilution series of 0.5 or greater
• Single concentration test
• Replicates
• Randomization (organisms/chambers)

21
Perspective is Everything
22
Test Conditions and Acceptability Criteria
23
Acute Toxicity Tests

• Test Procedures
• 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

24
Acute Test Acceptability Criteria

• Minimum control survival at least 90
• Temperature maintained _at_ 20 /- 1o C
• Maximum test organism age at start
• 14 days for fish
• 5 days for Mysid shrimp
• 24 hours for daphnids

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

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 maintained _at_ 25 /- 1o C
• Maximum test organism age at start
• 48 hours for fish
• 7 days for Mysid shrimp
• 24 hours for daphnids

27
Method Specific Test Conditions

• Test type and duration
• Temperature, light, DO, salinity
• Chamber size and volume
• Species selection, age and feeding

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

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

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

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.

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).

35
Chronic Test Data

• Average
• Effluent Mortality Dry weight w/Eggs
• 0 2.5 0.418 69.6
• 6.25 7.5 0.371 68.8
• 12.5 10.0 0.348 50.0
• 25.0 10.0 0.308 28.6
• 50.0 17.5 0.248 0.0
• 100.0 100.0 0.0 0.0
• NOEC 50.0 12.5 12.5
• IC25 55.7 23.2 10.7

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 (TUs)

• Reciprocal of the fractional LC50, NOEC, IC25
  value
• Calculated by dividing the value into 100
• TUa 100/LC50
• TUc 100/IC25

38
Standard Reference ToxicantProgram
39
Standard Reference Toxicants (SRTs)

• Purpose
• Frequency
• Acceptability Criteria
• Control Charts

40
Control Charts
41
Sample Collection
42
Subchapter 9

• N.J.A.C. 718 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

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.

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

47
Preservation

• For toxicity testing only temperature
  preservation permitted
• Refrigeration during sampling optional
• Refrigeration or icing immediately upon
  collection required

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

51
The Whole Effluent ApproachCapabilities

• 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

52
The Whole Effluent ApproachLimitations

• 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

53
Program Structure

• 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)

54
Laboratory Certification Program

• Regulations Governing the Certification of
  Laboratories and Environmental Measurements
  (N.J.A.C. 718)
• Subchapter 7 contains test methods
• Subchapter 9 contains the procedures governing
  sample collection and handling
• Formal certification program
• National and state programs

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

56
Acute Toxicity Methods

• Methods contained in rule at N.J.A.C. 718
• 5 concentrations control
• replicates
• 96 hours or less (species specific)
• Mortality or immobilization
• Receiving water for dilution preferred
• Test species

57
Chronic Toxicity Methods

• Incorporated by reference in N.J.A.C. 718-7.1(a)
• Part V includes additional requirements
• USEPA Methods (40 CFR 136)
• Certified laboratories
• Same test species as acute testing

58
Permitting
59
Limit Calculation

• N.J.A.C. 714A-13 - Effluent Limitations for DSW
  Permits
• USEPAs 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. 79-5.7(a)
  is an LC50gt50 effluent

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 acutechronic ratio

61
Limits - The Bottom Line

• Variability of data affects the final limit
  outcome
• The more data the better
• Ensure input values are appropriate

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

63
Toxicity Reduction Evaluations (TRE)

• Specific TRE language is included at N.J.A.C.
  714A-13.17(a)
• Language to exclude test results not considered
  representative included at N.J.A.C.
  714A-13.14(a)2
• Permittees responsibility
• NJDEP oversight role
• Series of stepped requirements
• Apply whether limit in effect or not

64
Regulatory Issues

• Recent Developments

65
USEPA Support for WET

• 1984 - EPA National Policy for WQBEL development
  for Toxic Pollutants
• 1989 - 40 CFR 122.44 Revised for WQBELs
• 1991 - Technical Support Document for Water
  Quality-based Toxics Control
• 1994 - WET Control Policy Updated
• 1995 - Incorporation of WET methods in 40 CFR 136

66
October 26, 1995

• 40 CFR 136.3 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

67
NJ WET Program History

• Early 1980s - Acute monitoring and limits used
  on a routine basis
• 1989 - Began use of chronic monitoring and
  chronic limits
• 1993 - Group permit challenge on chronic WET
• 1996 - Settlement and initial chronic WET program
  revisions
• 1997 - Final program revisions adopted

68
Settlement Agreement Requirements

• July 24, 1998
• Variability Guidance Document
• Method Guidance Document
• Interlaboratory Variability Study
• Rulemaking actions

69
Results

• 8 of 10 methods had test completion rates gt90
• 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 positiveslt5

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

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

73
Method Guidance Document

• July 28, 2000 (65 FR 46457)
• Minimum Significant Difference
• Confidence intervals
• Concentration response relationship
• Dilution series selection
• Dilution water selection

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.

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 laboratorys control charts
• Check test acceptability criteria
• Check sample holding times and Chain of custodys
• Obtain at leas 10 data points over gt1 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

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

81
Withdrawal of Two Methods

• Holmesimysis costata Acute Test
• west coast test organism
• Champia parvula Reproduction Test
• Methods can still be used

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

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

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

86
PMSD

• Percent Minimum Significant Difference
• Test Method Endpoint 10th PMSD 90th PMSD
• Fathead Minnow Growth 12 30
• C. dubia Reproduction 13 47
• Sheepshead minnow Growth (6.3) (23)
• Inland Silverside Growth 11 28
• Mysid Growth 11 37
• PMSD values calculated with Dunnetts test must
  be between within the range established by the
  10th and 90th PMSD values.

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)
• gt or 80 and lt or 100
• gt or 50 and lt 80
• gt10 and lt 50
• lt or 10

• Result Difference
• gt or 20
• gt or 15
• gt or 10
• gt or 9

92
Affirmative Defense

• N.J.A.C. 714-8.3 - 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
FAQs
95
Toxicity where you dont expect it??

• Stormwater
• Cooling Water
• Filter backwash
• High or low TDS
• Chlorine

96
Naturally low pH water

• N.J.A.C. 718-9.5(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?

• http//www.epa.gov/ecotox/
• 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

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.

101
WET Resources

• www.epa.gov/waterscience/WET
• www.epa.gov/ostwater/WET/index.html
• www.setac.org
• www.toxicity.com