whole effluent toxicity nov98

Standard Methods for Whole Effluent Toxicity Testing: Development and Application

NIWA Client Report: November 1998

Standard Methods for Whole Effluent Toxicity Testing: Development and Application Julie A Hall Lisa Golding Prepared for Ministry for the Environment Reproduction, adaptation, or issuing of this publication for educational or other non-commercial purposes is authorised without prior permission of the copyright holder(s). Reproduction, adaptation, or issuing of this publication for resale or other commercial purposes is prohibited without the prior permission of the copyright holder(s).

Reviewed by: Approved for release by: Burns Macaskill Bob Wilcock

NIWA Client Report: MfE80205 November 1998 National Institute of Water & Atmospheric Research Ltd PO Box 11-115, Hamilton New Zealand Tel: 07 856 7026 Fax: 07 856 0151

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CONTENTS 1.....Objective 1

2.....Introduction 1

3. Approach to development of Standard WETT protocols for routine use in New Zealand 8

4.....Development of standardised protocols with New Zealand native species 10

4.1 Marine Algae 12

4.2 Marine Fish 16

4.3 Summary of WETT with effluents using marine species 20

4.4 Summary of Marine Standard WETT protocols developed 21

4.5 Freshwater Invertebrates 21

4.6 Freshwater Fish 25

4.7 Summary of WETT with effluents using freshwater species 30

4.8 Summary of standardised freshwater WETT protocols developed. 30

4.9. Further development of WETT protocols 30

5.....Use of whole effluent toxicity tests 31

5.1 Use of WETT in Resource Consent Conditions 31

5.1.1 ... Current use of WETT in Resource Consents in New Zealand 32

5.1.2 .. Recommendations for use of WETT in Resource Consent Conditions 35

5.2 Use of WETT in Toxicity Identification Evaluations (TIE) 41

5.3 Use of WETT in Resource Consent Applications 42

6.....References 43

7.....Terminology 47

APPENDIX 1. Marine algae (Dunaliella tertiolecta) Chronic toxicity test

APPENDIX 2. Marine Sand dollar Embryo (Fellaster zelandiae)

Acute toxicity test

APPENDIX 3. Marine fish (Rhombosolea plebeia) Acute toxicity test

APPENDIX 4. Freshwater algae (Selenastrum capricornutum)

Chronic toxicity test

APPENDIX 5. Freshwater cladoceran (Cerodaphnia dubia) Acute toxicity test

APPENDIX 6. Freshwater amphipod (Paracalliope fluviatilis)

Acute toxicity test

APPENDIX 7. Freshwater fish (Gobiomorphus cotidianus) Acute toxicity test

Standard Methods for Whole Effluent Toxicity Testing: Development and Application iv

i

Executive Summary

Discharges into the aquatic environment of contaminated wastewater from industry, domestic sewage and stormwater, represent major sources of aquatic pollution. The complex nature of these wastewaters makes assessment of their potential biological impact difficult. Whole Effluent Toxicity Testing (WETT) has been widely used overseas to assess the potential toxicity of effluents. The results of these tests can be used for a variety of functions including resource consent monitoring and compliance, toxicity identification evaluations and evaluation of effluent treatment processes. Whole Effluent Toxicity Testing has been used in New Zealand since the mid 1980’s, generally using species imported from overseas or using New Zealand species with protocols developed for similar species overseas. The issues surrounding use of exotic species have been highlighted by recent amendments to the Biosecurity Act (1993). There is currently increasing pressure to use WETT in both effects and compliance monitoring conditions on resource consents. For effective use of WETT as conditions on resource consents it is important to have available a set of standardised protocols with New Zealand species which can be used throughout New Zealand for routine testing. The objective of this project was to develop standardised WETT protocols to meet this need. To complete an effective assessment of the potential impact of an effluent it is recommended that the effluent is tested with several species from different phylogenetic levels, for example an alga, an invertebrate and a fish. This project has developed standardised protocols for the freshwater invertebrates Ceriodaphnia dubia (water flea), and Paracalliope fluviatilis (amphipod), freshwater fish, Gobiomorphus cotidianus (common bully) and the marine alga Dunaliella tertiolecta and the marine fish Rhombosolea plebeia (sand flounder). Prior to this project protocols had been developed for the freshwater algae Selenastrum capricornutum and the marine invertebrate Fellaster zelandiae (sand dollar). This means there is now a set of both marine and freshwater, algal, invertebrate and fish standardised protocols for use with native New Zealand species. This report also highlights issues that need to be considered in the development of guidelines for use of WETT protocols in New Zealand.

Standard Methods for Whole Effluent Toxicity Testing: Development and Application 1

1. Objective

To develop standard protocols for Whole Effluent Toxicity Testing (WETT) using New Zealand native species.

2. Introduction

The discharge into the aquatic environment of contaminated wastewater from industry, domestic sewage, stormwater, pest and weed control, among others, represent major sources of pollution. The complex chemical nature of these wastewaters results from contributions from many diverse sources and the chemical complexity of such wastes makes assessment of their potential biological impact difficult.

To protect the aquatic environment from degradation due to these discharges the Resource Management Act (1991) seeks to safeguard the “life-supporting capacity of air, water, soil, and ecosystems” through sustainable management of natural and physical resources (S.5). To meet this requirement a regional council (or other consent authority) cannot grant a permit, or allow as a permitted activity, any discharge of wastewater which may have “any significant adverse effects on aquatic life” (s 70 and s 107). It is therefore essential that the techniques used to assess and monitor these discharges are sufficiently sensitive to detect potential toxic impacts.

There are three key methods for assessing the potential toxicity of an effluent:

i) quantification of the chemical components

ii) quantification of the biological toxicity of the effluent

iii) biological monitoring

Chemical testing involves detailed chemical analysis to characterise the wastewater and identify the contaminants. Reference can then be made to Water Quality Criteria, for example the ANZECC Water Quality Guidelines (ANZECC 1992) which give an acceptable concentration for the contaminants identified. This has the drawback of relying on toxicity data for individual chemicals, and therefore not accounting for synergistic, antagonistic or additive effects from a mixture of chemicals. It is very difficult to ensure that all components of a complex industrial or stormwater effluent are identified. The advantages and disadvantages of using chemical testing are outlined in Table 1.


Table 1. Advantages and disadvantages of the chemical-specific approach.

Advantages: • Treatment systems are more easily designed to meet chemical requirements

because of familiarity with these procedures. • The fate of a pollutant can be predicted through modelling. • Chemical analyses, in some cases, can be less expensive than toxicity testing.

Disadvantages: • All toxicants in complex wastewaters may not be known and, therefore, control

requirements for all potential toxicants may not be set. • It is not always clear which compounds are causing toxicity in the mixture. • Measurement of individual toxicants, particularly where many are present in the

mixture, can be expensive. • The bioavailability of the toxicants in the effluent is not assessed, and the

interactions between toxicants (e.g., additivity, synergism, antagonism) are not measured or accounted for.

Biological toxicity testing is a relatively simple laboratory bioassay procedure, and has the advantage of reflecting the combined toxicity of all chemicals contained in a complex effluent. The sampling for Whole Effluent Toxicity Testing (WETT) is conducted at the end of the pipe before the effluent is discharged into the receiving water. This allows the effects of the effluent to be isolated from any existing in-stream effects and also allows tests to be conducted with a range of effluent concentrations. When the potential toxic impact of an effluent on the biota of the receiving waters is assessed both the toxicity of the effluent and the dilution ratio of the effluent in the receiving waters must be considered. The advantages and disadvantages of WETT are outlined in Table 2.

Typically the effluent to be tested is collected and a series of dilutions prepared (e.g., 100%, 30%, 10%, 3% and 1% effluent), plus a control. The dilutions are usually prepared using a standard laboratory dilution water to ensure that the impact of only the effluent is being assessed. In site specific studies the receiving water can be used as the dilution water. Test organisms are placed in the various dilutions and incubated under controlled temperature and light conditions for a set period. After this exposure period, a measurement of toxic effect is made. The test endpoint may be one of many different variables (e.g., mortality, reduced fertilisation, lowered fecundity or reduced growth). The data can then be used to calculate that concentration which affects 50% of the test organisms i.e., an EC50 or LC50 or statistically derived thresholds like ‘no observed effect concentration’ (NOEC) and ‘lowest observed effect concentration’ (LOEC).


Table 2. Advantages and disadvantages of Whole Effluent Toxicity Testing approach.

Advantages: • The aggregate toxicity of all constituents in a complex effluent is measured. • The bioavailability of the toxic constituents is assessed, and the effects of

interactions between constituents are measured. • The concept is easily understood by the public, and provides tangible evidence of

environmental impact (or lack of it).

Disadvantages: • Properties of specific chemicals (such as potential for bioaccumulation) are not

assessed. • There is no identification of specific toxic components. This problem can be

addressed by using Toxicity Identification Evaluations (TIE) which use WETT protocols to identify at least the category of toxic chemicals.

• Toxicity of contaminants where there are chemical\physical conditions present (e.g., pH changes, salinity changes, photolysis) that act on toxicants in such a way as to “release” toxicity downstream will not be measured.

Biological monitoring can also be used to assess the impact of toxic substances on the ecosystem of the receiving water. Biological monitoring involves surveys of the receiving water biota to assess if there are any changes in community structure or function in the ecosystem. The advantages and disadvantages of this approach are outlined in Table 3.


Table 3. Advantages and disadvantages of WETT compared to biological monitoring.

Advantages:

• Testing over a range of effluent dilutions allows the effect level to be identified. • Dilution of the effluent with the dilution water means that the measured effects are

attributable to that effluent discharge. • Establishes magnitude of toxic risk. • Rigorous testing protocol provides the ability for routine compliance monitoring. • Testing may be used to track the source of the contaminants with “up-the-pipe”

sampling using Toxicity Identification Evaluations (TIE). • Demonstration of a toxic effect with the selected test species and ambient dilution

may indicate impacts on local species.

Disadvantages: • Demonstrating no measurable toxicity may not absolutely preclude the absence of

environmental impact, because laboratory tests do not include all species present in the local environment.

• Laboratory tests may not integrate other stress factors operating on local communities, particularly those operating over long time periods.

• Laboratory tests cannot easily be used to predict community level ecological impacts.

In situations where a high environmental risk is involved it may be appropriate to use all three approaches to determine the impacts of contaminants on an ecosystem. This integrated approach offers advantages to dischargers in not requiring unnecessarily harsh effluent standards, and environmental advantages of integrating all potential contaminant effects. Use of this approach strengthens the conclusion that can be drawn regarding the potential impacts of toxic contaminants. For example if only WETT is used, the potential impact of bioaccumulated chemicals, low concentrations of persistent chemicals, and species specific chemicals (e.g., some pesticides) may not be adequately assessed.

While there are obvious differences between toxicity assessments based on chemical measurements compared with tests on whole effluent, it is important to appreciate that chemical criteria themselves are derived from biological toxicity testing. The USEPA recommendation is that because the whole-effluent and chemical-specific approaches are largely complementary both should be used (USEPA 1989), with biological monitoring also being used in some situations.


In recent years the availability of standardised protocols for WETT and their use for routine assessment of complex industrial wastes have increased substantially overseas. This progress followed the USEPA’s change to the policy regulating toxic materials in effluents through the National Pollutant Discharge Elimination System (NPDES), in March 1984. This change allowed for the use of biological testing of effluents as a regulatory tool in NPDES permits. In several countries toxicity testing is now carried out routinely using standardised test protocols. Examples of standard toxicity tests which are currently being used for water column toxicity assessment in the USA, Australia and Canada are given in Tables 4 and 5.


Table 4. Examples of standard protocols used overseas for WETT with freshwater species.

Group Test Type Species Reference

Bacteria Microtox Photobacterium phosphoreum Beckman 1982, Environment Canada 1992b.

Algae Growth inhibition Selenastrum capricornutum Environment Canada 1992a, ASTM 1997.

Invertebrates Survival Daphnia magna

Daphnia pulex

USEPA 1993, Environment Canada 1990c,

ASTM 1997.

Survival, Reproduction Ceriodaphnia dubia USEPA 1993, Environment Canada 1997b.

Fish Survival Oncorhynchus mykiss USEPA 1993, Environment Canada 1990a,b,

1992c.

Survival Cyprinodon variegatus USEPA 1993

Survival Pimephales promelus USEPA 1993

Survival Gasteroteus aculeatus Environment Canada 1990d.


Table 5. Examples of standard protocols used overseas for WETT with marine species.

Group Test type Species Reference Bacteria Microtox Photobacterium phosphoreum Beckman 1982, Environment Canada 1992b. Algae Growth inhibition Minutocellus polymorphus Walsh 1987 Embryo development Champia parvula ASTM 1997 Embryo development Fucus edentatus ASTM 1997 Embryo development Lamineria saccharina ASTM 1997 Embryo development Macrocystis pyrifera USEPA 1995b, ASTM 1997 Fertilisation Hormosira sp. Gunthorpe et al. 1997a,b Invertebrates Echinoids/Urchins Embryo development Arbacia punctulata USEPA 1995b, ASTM 1997 Embryo development Strongylocentrotus droebachiensis ASTM 1997 Embryo development Strongylocentrotus purpuratas USEPA 1995b, ASTM 1997 Embryo development Dendraster excentrius USEPA 1995b, ASTM 1997 Shrimp Survival Mysidopsis bahia USEPA 1993 Survival Holmesismysis costata Anderson et al. 1990, ASTM 1997, USEPA

1995b Molluscs Embryo development Crassostrea gigas USEPA 1995b, ASTM 1997 Embryo development Crassostrea virginica Embryo development Mercenaria mercenaria Larval development Mytilis edulis USEPA 1995b Larval development Haliotis rufescens USEPA 1995b Fish Survival and growth Atherinops affinis USEPA 1995b Survival Mendia beryllina USEPA 1993 Survival Mendia menidia USEPA 1993 Survival Mendia peninsulae USEPA 1993


The tests listed do not include all tests developed as many test species do not meet criteria for routine use. These criteria generally include ecological importance, wide distribution and abundance, culturability or year round availability, taxonomically distinct and well described.

WETT can be used in a variety of ways in the management of wastewater discharges. For example in

(i) Resource Consent conditions

(ii) Resource Consent applications

(iii) Toxicity Identification Evaluation (TIE).

(iv) Evaluation of effluent treatment processes

(v) Multiple wasteload and waste assimilation studies

The objective of this project was to develop standardised WETT protocols with native species which can be used widely throughout New Zealand for the routine assessment of effluent toxicity.

3. APPROACH TO DEVELOPMENT OF STANDARD WETT PROTOCOLS FOR ROUTINE USE IN NEW ZEALAND

The use of WETT measurements in a routine monitoring programme and also potentially as a compliance measurement on a resource consent means that routine test performance is important. This requires the protocols to be clear and extensive and apply to species that are widely available in New Zealand. The factors which have been identified for standardisation in WETT protocols are outlined in Table 6.


Table 6. Factors for standardisation in WETT protocols.

Test Parameter

Test Organism:

Source:

Test Type:

Test duration:

Temperature:

Light intensity/quality:

Test chambers:

Test solution volume:

Dilution water:

Renewal of test concentrations:

Age of test organisms:

Number of concentrations:

Number of test organisms per chamber:

Number of replicate chambers per treatment:

Aeration:

Feeding regime:

Observation:

Chemical measurements :

Endpoint:

Test acceptability criteria:

To provide a useful tool for use by resource managers and dischargers (in New Zealand), standard WETT protocols were required for freshwater and marine algae, invertebrates and fish. The use of three phylogenetically different species is strongly recommended when testing an effluent as it provides the greatest confidence in detecting potentially adverse impacts of an effluent (USEPA 1991b). There is no single most sensitive species or group. Schimmel et al. (1989) showed, in a series of effluent tests with five species and three phylogenetic levels, that no single species was consistently the most sensitive to all the effluents tested.

In developing WETT protocols for use in New Zealand there are several reasons for using native New Zealand species. The first reason is that some native species have been shown to be more sensitive to toxicants than test species commonly used overseas. For example, Hickey (1989) showed that the New Zealand strain of Ceriodaphnia dubia was more sensitive than Daphnia magna (used widely overseas


as a standard species for toxicity testing) by a factor of up to 4 in acute tests with a number of reference toxicants and effluents. However, in chronic tests no differences in sensitivity were observed. Native stream invertebrates were also shown to be very sensitive and Hickey and Vickers (1994) suggested that they may not be adequately protected by the USEPA criterion for ammonia based on salmonid sensitivity. The second reason is that use of native species is much more easily defended, particularly to the public. A third reason is that careful consideration should be made of the environmental risk of importation and use of exotic species. This has been highlighted by the recent amendments (1997) to the Biosecurity Act (1993) which has placed tighter controls on the import and use of exotic species in New Zealand.

There are two basic types of toxicity test, acute and chronic. An acute test measures the deleterious effect on an organism over a short term exposure relative to the life span of the organism. These tests usually run for 24 to 96 hours. In acute tests, the most common end point measured is mortality (Rand & Petrocelli 1985) with the results generally reported as % mortality at a given concentration or an LC50. Chronic tests are designed to identify the concentration of a chemical or effluent that will interfere with normal growth and development or attainment of reproduction potential. These tests are generally more sensitive than acute tests and can have a range of end points. For example, growth, number or % of embryos to complete development, or the number of larvae that survive and grow normally (Rand & Petrocelli 1985).

Following the lead of the USEPA, this project focused on developing standardised protocols for acute toxicity tests with New Zealand native invertebrates and fish and chronic growth tests with algae. In future development should focus on chronic protocols for invertebrates and fish.

4. DEVELOPMENT OF STANDARDISED PROTOCOLS WITH NEW ZEALAND NATIVE SPECIES

Prior to the initiation of this project standardised protocols had been developed for the freshwater alga Selenastrum capricornutum (Appendix 4) and the marine invertebrate Fellaster zelandiae (sand dollar). S. capricornutum which occurs in New Zealand is widely used as a standard test species in a number of countries including Canada and the standardised protocol had been adapted from the Environment Canada microplate algal growth protocol (Environment Canada 1992a and 1997e). The standardised protocol for the sand dollar (Appendix 2) is a test based on the fertilisation assay for echinoids (Environment Canada 1992d and 1997d).


Building on these methods this project focused on developing standardised WETT protocols for marine algae and fish and freshwater invertebrates and fish. A standard approach was used. Initially a range of species was identified as suitable candidates for consideration. The factors considered in choosing these species were:

• ecological importance; • wide distribution and abundance; • relevance to New Zealand culturally, recreationally or economically; • potentially culturable or available for field collection year round; • taxonomically distinct and well described; • toxicity testing protocols for similar species existed elsewhere.

After a number of species were identified as suitable candidates initial screening tests were conducted using four reference toxicants to assess the potential sensitivity of the species. The four reference toxicants used were the metals Zn and Cd and two organic compounds, sodium dodecyl sulphate (SDS) and phenol. The use of reference toxicants allows comparison of sensitivity between species and also evaluation of a test stability and precision over time (Environment Canada 1990e).

Once the initial screening tests had been completed for each group of organisms a further assessment was conducted to select species suitable for development of a standardised WETT protocol. The criteria used in this evaluation were:

• tolerance of being handled under laboratory conditions; • sensitivity to toxicants; • potential to develop protocols for chronic toxicity test;

Once a species had been identified for further work, appropriate culture methods were refined if appropriate and standardisation of the protocol was undertaken. The key issues in the protocol standardisation were:

• consistency of responses to toxicants; • species sensitivity; • seasonal variability particularly in field collected species.


4.1 Marine Algae

The use of both macro and microalgae was explored for the development of a standardised marine algal test. Two species of macro algae were investigated. The macro alga Hormosira banksii (Neptune’s Necklace) has been used in Australia for the assessment of effluent toxicity using a fertilisation test (Gunthorpe et al. 1997a,b). Extensive work was conducted with H. banksii to evaluate this method. Inconsistency in spawning this species meant that this method was not suitable for standardisation. The second macro alga investigated was Ulva fasciata (sea lettuce). A method based on an endpoint of zoospore germination and growth (Hooten & Carr 1998) was evaluated. Difficulty in consistently inducing zoospore release led to this method also being abandoned. This problem had been identified by the originators of the method (Carr pers com).

Five microalgae species Thalassiosira pseudonana, Dunaliella tertiolecta, Tetraselmis sp., Pavlova sp., and Nitzschia pungens (non-toxic form), were evaluated for use in a chronic growth inhibition test. These are all species that occur in New Zealand waters and are available as axenic cultures from the CSIRO Culture Collection in Hobart or in the case of Nitzschia pungens from the Cawthron Institute culture collection. The basic method assessed was the algal growth microplate method (Environment Canada 1992a). Initial growth tests showed that T. pseudonana and Pavlova sp. tended to have variable growth in the plates and Nitzschia pungens tended to stick to the microplates. In view of these problems these three species were not considered for further investigation.

Screening tests with the four reference toxicants were conducted with D. tertiolecta and Tetraselmis sp. Dunaliella tertiolecta was the more sensitive of the two species overall (Table 7).

Other than the difference in sensitivity, the two species performed in a similar manner and hence D. tertiolecta was chosen for development of a standardised protocol for marine algae (Appendix 1). Results of the protocol standardisation procedures showed that D. tertiolecta had moderate precision with the reference toxicant Zn (Fig. 1), with a coefficient of variation (CV) of the Zn EC50 value over all tests conducted of 67% (Table 8). This coefficient of variation is high, however it is similar to the CV of 56% recorded for Minutocellus polymorphus, a species frequently used for testing both in New Zealand and overseas. The high CV values for both species may be a result of the use of differences in the seawater source used. The CV for tests conducted with the reference toxicant SDS with D. tertiolecta was 17%.


The median MSD (Minimum Significant Difference), which measures the difference that could be detected between the control and treatment data, was 12% for tests with the reference toxicant Zn and 24% with effluents (Table 8). This suggests that the protocol is reasonably robust. If smaller differences need to be detected the protocol would need to be modified by increasing the number of replicates.

Table 7. Sensitivity of marine micro algae to four reference toxicants from initial screening tests

Species Toxicant EC50

(mgL-1) 95% CI

Tetraselmis sp. Zn 0.71 NC

Dunaliella tertiolecta Zn 0.25 0.0-0.42

Tetraselmis sp Cd 17.1 9.9-22.3

D. tertiolecta Cd 1.35 1.2-1.6

Tetraselmis sp. Phenol 44.9 34.5-51.5

D. tertiolecta Phenol 69.9 62.2-77.8

Tetraselmis sp. SDS 8.14 5.4-9.2

D. tertiolecta SDS 1.2 1.1-1.4


Table 8. Summary of test performance

Species Common Name

Test Mean Control Survival

Mean EC50 Zn mgL-1

Coefficient Variation Zn EC50 (%)

Median MSD For Zn tests (%)

Mean EC50 SDS mgL-1

Coefficient Variation SDS EC50 (%)

Median MSD for effluent tests (%)

Marine Dunalliella tertiolecta

Microalgae Chronic Growth Test

N/A 0.27 67 12 6.6 17 24*

Fellaster zelandiae

Sand dollar Acute (36 hr) embryo development

N/A 0.08 38 8 - - 7

Rhombosolea plebeia

Sand flounder Acute (96 hr) lethality test

98% 2.8 30 24* 4.4 6.9 35*

Freshwater Selenastrum capricornutum

Microalgae Chronic Growth Test

N/A 0.009 38 13 - - 14

Ceriodaphnia dubia

Water flea Acute (48 hr) lethality test

94.7 0.36 33 21 15.3 16.3 19

Paracalliope fluviatilis

Amphipod Acute (48 hr) lethality test

89.5 0.54 58 18 32.9 43.9 21

Gobiomorphus cotidianus

Common bully Acute (96 hr) lethality test

99.5 3.6 27.9 8* 43.0 23.3 29

* less than 10 tests


Mean

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-2 SD

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+2 SD

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EC50

(mg/

L)

Figure 1. Quality Control chart for Zn EC50 for D. tertiolecta growth test.

Comparison of the sensitivity of D. tertiolecta and the international benchmark species Minutocellus polymorphus, showed that M. polymorphus was an order of magnitude more sensitive to Zn and phenol, however both species had a similar sensitivity to Cd, SDS and phenol (Table 9).

Table 9. Mean sensitivity (EC50) of D. tertiolecta and. M. polymorphus to Zn, CD, SDS and phenol from standardised testing

EC50(mg L-1) ± Standard Deviation Species Zn Cd SDS Phenol

D. tertiolecta 0.27±0.18 0.49±0.14 6.6±1.1 149±110

M. polymorphus 0.02±0.01 0.56±0.12 2.4±0.6 123±32

D. tertiolecta was relatively sensitive when compared to growth inhibition tests of overseas marine algal species to Zn which had EC50 values ranging from 0.19 to 101 mg L-1 (USEPA 1987).

The evaluation of sensitivity with this species and others throughout this report has been completed using Zn. This limits the conclusions that can be drawn as it represents only one toxicant, however Zn is one of the few reference toxicants where there is an extensive database that can be used for comparison of a wide range of species. Although M. polymorphus is more sensitive to some toxicants it is not an


appropriate species for a standardised protocol for routine use in New Zealand, as the 1997 amendments to the Biosecurity Act (1993) mean that use of this species would currently be restricted to laboratories that are registered as Transitional Facilities under the Act.

D. tertiolecta is an appropriate species for routine use in WETT procedures in New Zealand, having met a significant number of the criteria for a tests species.

4.2 Marine Fish

Three fish species Rhombosolea plebeia (sand flounder), Peltorhamphus latus (common sole) and Arripis trutta (kahawai) were selected for initial screening with the four reference toxicants. The acute lethality test used was based on the Environment Canada Acute Lethality Test using Rainbow Trout (Environment Canada 1990 a, b). Sole and kahawai had similar sensitivities to Zn and Cd while the kahawai was the most sensitive to the organic toxicants SDS and phenol (Table 10). However, the EC50 for both species was similar. As both species showed good control survival under laboratory conditions and the sole could be collected year round compared to the seasonal availability of kahawai, sole was chosen for protocol standardisation.

D. tertiolecta

• ecological importance ?

• wide distribution and abundance ?

• relevance to New Zealand culturally, recreationally or economically N/A

• potentially culturable or available for field collection year round **

• taxonomically distinct and well described **

• toxicity testing protocols for similar species existed elsewhere **

• tolerance of being handled in the laboratory and laboratory conditions **

• consistency of responses to toxicants *

• sensitivity to toxicants *

* good, ** very good, ? unknown


Table 10. Sensitivity (96-hour EC50) of marine fish to reference toxicants zinc, cadmium, SDS and phenol, from initial screening tests.

Species Toxicant EC50 (mg L-1)

95% CI

Mean wet weight

(g)

Mean length (mm)

P. latus

(Sole)

Zinc 1.83 (1.32-2.52) 0.43 34.9

A. trutta

(Kahawai)

Zinc 2.65 (2.00-3.49) 1.91 48.9

R. plebeia

(Sand flounder)

Zinc 12.9** (8.1-400) 0.51 33.7

P. latus Cadmium 0.28 (0.13-0.43) 0.43 34.9

A. trutta Cadmium ∼ 0.4 NR 1.91 48.9

R. plebeia Cadmium ND ND 0.51 33.7

P. latus SDS 3.88 * NR 0.40 35.9

A. trutta SDS 1.51 (1.30-1.56) 1.91 48.9

R. plebeia SDS 4.22 (4.07-4.39) 0.31 27.3

P. latus Phenol 10.9 (9.87-12.04) 0.93 50.7

A. trutta Phenol 7.63 (6.92-8.41) 1.91 48.9

R. plebeia Phenol 9.38 (8.45-10.41) 0.77 36.5

* mortality at highest test concentration (1.8 mg L-1) was 10%

** note this is result of initial screening test and is higher than mean EC50 established after

further testing

ND = not determined

NR = not reliable


After initial tests with sole were conducted, a significant increase in the size of the fish was observed. This may affect the sensitivity of the fish and also made handling the fish in the laboratory difficult. It was decided to continue the standardisation of the protocol with the sand flounder which has been shown to have a much longer spawning period and hence longer period when similar sized fish would be available for testing. The sand flounder showed good control survival with a mean control survival of 98%, however restricted testing does not allow a clear conclusion regarding any seasonal variability in sensitivity to either Zn (Fig. 2) or SDS. The test precision with sand flounder was good with a test CV of the EC50 of 30% for Zn and 7% for SDS.

The median MSD was 24% for Zn and 35% for the effluents tested (Table 8). The high value particularly for the effluent testing may in part be due to the low number of tests completed.

Mean

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1.4

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3.4

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4.4

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Date

EC50

(mg/

L)

Figure 2. Quality Control chart for Zn EC50 for R. plebeia 96-h survival test.

When the sensitivity to Zn is considered in comparison to species tested elsewhere (Fig. 3) it lies in the upper 50% in terms of sensitivity, suggesting that use of these species as a standard test species would provide protection for only approximately 50% of the species. The use of kahawai or sole would produce similar results (Fig. 3).


0

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0.01 0.1 1.0 10 100 1000Species mean acute EC50 values (mg L

Perc

enta

ge o

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NZ Sand Flounder R. plebeia

NZ KahawaiA. trutta

NZ SoleP. latus

Figure 3. Cumulative plot of acute Zn EC50 data for marine species (USEPA 1987) used to derive water quality criteria with marine fish, test results marked.

There is a need for further work to be conducted to establish if more sensitive marine fish species occur in New Zealand. This would establish if the results from WETT with R. plebeia will provide protection for a significant number of native species. The three species tested in this study had similar sensitivities to Zn, SDS and phenol with R. plebeia being the least sensitive to Cd. Until this work can be conducted R. plebeia is an appropriate species for routine use in WETT procedures in New Zealand, having met a significant number of the criteria for a test species.


4.3 Summary of WETT with effluents using marine species

To evaluate the protocols developed for use with complex effluents, tests were conducted with 4 different effluents. Effluents 1 and 4 were municipal sewage, 2 was a landfill leachate and 3 was an industrial effluent.

The testing showed that D. tertiolecta and R. plebeia had similar responses to effluent 2 and that R. plebeia was more sensitive to effluent 3 (Table 11). These results show the differences in sensitivity to effluents compared to reference toxicants. On the basis of the reference toxicant results D. tertiolecta would have been expected to be the more sensitive of the two species. These results highlight the need to consider a range of species from different phylogenetic levels when evaluating the toxicity of an effluent.

Table 11. Sensitivity of marine species to effluents. EC50 as % of effluent with 95% Confidence Interval.

Effluent 1 2 3 4

D. tertiolecta - 5.1(4.0-5.8) 11.5(10.5-12.3) >32

R. plebia 44.6(29.7-73.0) 3.0(0.7-4.6) 2.5(2.1-2.9) 72.4(68.0-77.0)

R. plebeia

• ecological importance * *

• wide distribution and abundance *

• relevance to New Zealand culturally, recreationally or economically **

• potentially culturable or available for field collection year round *




• consistency of responses to toxicants **

• sensitivity to toxicants <*

* good, ** very good,


4.4 Summary of Marine Standard WETT protocols developed

Standard protocols have been developed for a chronic growth inhibition test with the alga D. tertiolecta (Appendix 1), a short term chronic embryo development test with the sand dollar F. zelandiae (Appendix 2) and an acute lethality test with the sand flounder R. plebeia (Appendix 3). The alga and the sand dollar have been shown to have a sensitivity to Zn in the lower 25% of marine organisms tested (USEPA 1987). Use of these as standard test species is likely to provide a high level of protection for marine ecosystems. The sand flounder can be used as a routine test species. It was not particularly sensitive to the reference toxicants, however it showed a similar or greater sensitivity than D. tertiolecta to two effluents. Further work is required to establish the sensitivity of other native marine fish species, relative to this species.

4.5 Freshwater Invertebrates

Six freshwater invertebrate species were chosen for initial screening on the basis of data from Quinn & Hickey (1990) who conducted a survey of New Zealand streams and rivers and reported the abundance and ecological distribution of a large number of invertebrates. All the test organisms were collected from water bodies in the Waikato region. Results of the initial screening (Table 12) showed some species such as Deleatidium sp. were very sensitive but control mortality rates were unacceptably high during these tests. This may have been due to thermal shock and there is ongoing work on the thermal sensitivity of these organisms.

Ceriodaphnia dubia (water flea) was selected for protocol standardisation using an acute lethality test. C. dubia had high control survival, high sensitivity, can be cultured and is used overseas as a standard test organism (USEPA 1993). As C. dubia is essentially an inhabitant of lake and pond waters it is desirable to also have a freshwater invertebrate species from a stream ecosystem. Based on sensitivity to reference toxicants, control survival and year-round abundance Paracalliope fluviatilis (amphipod) was selected as an optional test species for development of an acute lethality test. Both C. dubia and P. fluviatilis were good choices for protocol standardisation as there is already a chronic test developed with C. dubia (Environment Canada 1997a) and there is a chronic amphipod protocol for very similar species (Environment Canada 1997b).


Table 12. Sensitivity (96-hour EC50) for native freshwater invertebrates to cadmium, zinc, SDS and phenol in initial screening tests


(mg L-1)

95% CI* Exposure (hours)

% control mortalit

y Ceriodaphnia dubia (Cladoceran)

Cadmium 0.11 0.10-0.12 48 0

Paracalliope fluviatilis (Amphipod)

0.07 0.04-0.11 96 22

Deleatidium spp (Mayfly)

0.57 0.28-8.32 96 2

Potamopyrgus antipodarum (Snail)

0.72 0.62-0.82 96 0

Coloburiscus humeralis (Mayfly)

ND* ND ND ND

Olinga feredayi (Caddisfly)

>10 ND 96 0

Pycnocentria evecta (Caddisfly)

>10 ND 96 0

Paratya curvirostris (Shrimp)

0.18 0.11-0.28 96 0

Ceriodaphnia dubia Zinc 0.50 0.43-0.58 48 0 Paracalliope fluviatilis 0.58 NR* 96 22 Deleatidium spp 18.5 15.6-22.0 96 43 Potamopyrgus antipodarum 11.2 8.4-20.6 96 0 Coloburiscus humeralis ND ND ND ND Olinga feredayi >10 ND 96 0 Pycnocentria evecta >10 ND 96 0 Paratya curvirostris 14.0 9.8-20.1 96 0 Ceriodaphnia dubia Phenol 20.2 17.4-23.4 48 7 Paracalliope fluviatilis 33.2 28.3-38.9 96 22 Deleatidium spp 20.1 16.9-23.9 96 2 Potamopyrgus antipodarum 119.6 106.8-133.9 96 0 Coloburiscus humeralis 11.0 NR 96 25 Olinga feredayi ND ND ND ND Pycnocentria evecta >100 ND 96 0 Paratya curvirostris 22.9 21.4-24.4 96 0 Ceriodaphnia dubia SDS 15.8 13.3-18.8 48 7 Paracalliope fluviatilis 37.0 30.2-45.5 96 22 Deleatidium spp 25.6 20.8-31.4 96 2 Potamopyrgus antipodarum 54.1 48.9-59.9 96 0 Coloburiscus humeralis 37.3 32.1-43.3 96 25 Olinga feredayi ND ND ND ND Pycnocentria evecta >100 ND 96 0 Paratya curvirostris 189.5 152.7-315.0 96 0 * CI = confidence interval; ND = not determined; NR = not reliable


Optimisation of culture conditions for C. dubia was difficult. The species is very sensitive to temperature fluctuations, vibration and food supply. Once the culture method for C. dubia was established, tests using Zn as the reference toxicant were used to investigate sensitivity.

Some test protocols for C. dubia recommend the use of only certain broods (2nd, 3rd) of C. dubia for toxicity testing. Results in this study show that the sensitivity of different broods to toxicants was not significantly different. Therefore, the offspring of C. Dubia of varied ages and producing different broods can be pooled together when running a test.

Standardisation of the P. fluviatilis test proved difficult with low control survival being an intermittent problem. Two approaches were used to overcome this, one was to use similar sized organisms in the test to avoid cannibalism and the second was the use of cotton gauze in the containers to provide a substrate for the organisms.

Both C. dubia and P. fluviatilis showed good control survival with means of 95%, and 90% respectively. C dubia showed good test precision (Fig. 4) with test CV’s of 33% and 16% for Zn and SDS respectively. However P. fluviatilis showed higher test variability (Fig 5) with test CV’s of 58 and 44% for Zn and SDS tests respectively (Table 8). This may have been due to a number of factors such as variability in condition of field collected animals, variability in organism size or a lack of substrate in test containers. The MSD values for both Zn and effluents for C. dubia and P. fluviatilis were 18-21% (Table 8) indicating that these tests are reasonably robust. The sensitivity of both species was high with both species in the lower 25% for Zn sensitivity (Fig 6).

Mean

-1 SD

-2 SD

+1 SD

+2 SD

0.05

0.15

0.25

0.35

0.45

0.55

0.65

0.75

0.85

05/2

9/96

01/1

3/97

01/1

5/97

01/2

0/97

02/0

5/97

02/0

7/97

02/1

0/97

02/1

0/97

02/1

2/97

04/0

9/97

05/1

2/97

05/3

1/97

06/0

9/97

10/0

1/97

11/1

2/97

06/1

8/98

12/0

2/98

Test Date

EC50

(mg/

L)

Figure 4. Quality Control chart for Zn EC50 for C. dubia 48 h survival test.


Mean

-1 SD

-2 SD

+1 SD

+2 SD

0

0.2

0.4

0.6

0.8

1

1.2

02/1

0/97

07/2

1/97

09/0

8/97

11/1

3/97

11/2

5/97

12/0

1/97

02/1

8/98

03/1

3/98

06/1

5/98

Test Date

EC50

(mg/

L)

Figure 5. Quality Control chart for Zn EC50 for P. fluviatilis 48-h survival test.

0

20

40

60

80

100

0.01 0.1 1.0 10 100

Per

cent

age

of s

peci

es te

sted

Species mean acute EC50 values (mg L-1)

NZ AmphipodP. fluviatilis

NZ WaterfleaC. dubia

Figure 6. Cumulative plot of acute Zn EC50 data for freshwater species (USEPA 1987) used

to calculate water quality criteria, test results marked.


C. dubia and P. fluviatilis are appropriate species for routine use in WETT procedures in New Zealand with both species having met a significant number of the criteria for a test species. Standardised protocols are provided in Appendices 5 and 6.

4.6 Freshwater Fish

Screening tests were conducted on six species of freshwater fish considered to be suitable for use in acute lethality tests. Based on sensitivity (Table 13) and year-round availability the native smelt Retropinna reptropinna was selected for protocol standardisation with an acute lethality test, based on the Environment Canada Acute Lethality Test using Rainbow trout (Environment Canada 1990 a, b). Whitebait may

C. dubia


• wide distribution and abundance **





• tolerance of being handled in the laboratory and laboratory conditions *


• sensitivity to toxicants **

* good, ** very good

P. fluviatilis










*good, ** very good,


have been more sensitive than smelt but results of the tests with Zn and Cd were affected by the presence of salt water in the tests which was present to reduce fungal infections. Detailed experimental work with the smelt showed this species to be very sensitive to handling during collection and acclimation, resulting in high control mortality during the tests. As a result the common bully Gobiomorphus cotidianus was used for the development of the standardised protocol. The bully showed no apparent seasonal pattern in sensitivity and had good test precision with Zn (Fig. 7) and SDS with test CV’s of 28 and 23% respectively, had high control survival (mean 99%) and was moderately sensitive (Fig. 8). The MSD values for both Zn and effluents were above 25% (Table 8) indicating this protocol may not be particularly robust. Further tests need to be conducted. The EC50 of the common bully for Zn lies in the lower 50% but they are less sensitive than Rainbow trout (Oncorhynchus mykiss) (Fig. 8). Rainbow trout could certainly be used as an alternative species for testing in New Zealand as it is present in New Zealand waters, is sensitive to toxicants, falls in the lower 25% and is more sensitive than any of the native species tested (Table 13, Fig. 8). Rainbow trout are also relevant to New Zealand being identified for protection in the Resource Management Act (1991).


Table 13. Sensitivity (96-hour EC50) of native freshwater fish to zinc, cadmium, SDS and phenol in initial screening tests.


(mg L-1) Confidence

Interval Mean

Size±±±±SD (mm)

Mean Wet Weight±±±±SD

(g) R. retropinna (smelt)

Zinc 1.45 1.21-1.73 62.6±6.5 1.88±0.81

G. cotidianus (bully)

2.14 1.82-2.51 33.6±5.7 0.46±0.21

A. dieffenbachii (long fin eel)

8.92 7.71-10.31 108.5±7.8 1.31±0.34

A. australis (short fin eel)

11.13 9.28-13.35 95.8±11.2 0.84±0.29

O. mykiss (Rainbow trout)

0.29 0.25-0.33 34±2 0.29±0.06

G. maculatus (Whitebaite)

13.7 12.6-13.9 49±2 0.38±0.07

R. retropinna

Cadmium 0.63 0.39-0.81 68.1±8.7 2.47±1.23

G. cotidianus

1.85 1.57-2.19 30.6±6.6 0.36±0.25

A. dieffenbachii

3.57 3.20-3.98 110.1±7.8 1.31±0.34

A. australis

8.72 7.49-10.15 95.8±11.2 0.84±0.29

O. mykiss 0.003 NR 34±2 0.29±0.06 G. maculatus 21.1 18.8-23.8 49±2 0.38±0.07 R. retropinna

SDS 28.27 24.81-32.21 68.1±8.7 2.47±1.23

G. cotidianus

43.08 NR 30.6±6.6 0.36±0.25

A. dieffenbachii

44.07 42.15-46.09 110.1±7.7 1.27±0.34

A. australis

47.03 42.35-52.22 94.0±10.6 0.72±0.28

O. mykiss 37.59 34.23-41.28 33±3 0.31±0.07 G. maculatus 24.0 NR 49±2 0.38±0.07 R. retropinna

Phenol 4.48 3.83-5.25 68.1±8.7 2.47±1.23

G. cotidianus

14.46 13.27-15.75 30.6±6.6 0.36±0.25

A. dieffenbachii

24 22.48-25.62 110.1±7.7 1.27±0.34

A. australis

21.46 NR 94.0±10.6 0.72±0.28

O. mykiss 4.60 3.66-5.80 33±3 0.31±0.07 G. maculatus 9.02 8.21±9.92 49±2 0.38±0.07

NR = not reliable


Mean

-1 SD

-2 SD

+1 SD

+2 SD

1

2

3

4

5

6

04/2

1/97

06/0

9/97

08/1

1/97

11/1

0/97

12/1

5/97

02/1

4/98

06/1

5/98

Date

EC50

(mg/

L)

Figure 7. Quality Control chart for Zn EC50 for G. cotidianus 96-h survival test.

0

20

40

60

80

100

0.01 0.1 1.0 10 100

Per

cent

age

of s

peci

es te

sted

Species mean acute EC50 values (mg L-1)

NZ BullyG. cotidianus

Rainbow TroutO. mykiss

Figure 8. Cumulative plot of acute Zn EC50 data for freshwater species (USEPA 1987) used to develop water quality criteria freshwater species, test results marked.


The standardised WETT protocol for G. cotidianus is appropriate for routine use in New Zealand having met a significant number of the criteria for a test species. However O. mykiss could also be used as a routine test species using the Environment Canada protocols (Environment Canada 1990a,b, 1992c).

G. cotidianus



• relevance to New Zealand culturally, recreationally or economically *

• potentially culturable or available for field collection year round **





• sensitivity to toxicants <*


O. mykiss

• ecological importance **


• relevance to New Zealand culturally, recreationally or economically **





• consistency of responses to toxicants not tested




4.7 Summary of WETT with effluents using freshwater species

The same four effluents that were used with the marine species (Section 4.3) were used to evaluate the protocols developed for C. dubia, P. fluviatilis and G. cotidianus. For the three effluents where results were obtained for all species P. fluviatilis was the most sensitive for two out of the three effluents, with C. dubia and G. cotidanus having similar sensitivities (Table 14). For the third effluent C. dubia was significantly more sensitive than either P. fluviatilis and G. cotidianus which had similar sensitivities. These results highlight the need to consider a range of species from different phylogenetic levels when evaluating the toxicity of an effluent.

Table 14. Sensitivity of freshwater species to effluents. EC50 as % of effluent with 95% Confidence Interval

Effluent Species 1 2 3 4

C. dubia 45(35-37) 3.0(1.5-5.8) test not valid 18(11.4-26.6)

P. fluviatilis 9.2(6.1-13.9) 0.4(0.19-0.63) 3.3(2.5-4.0) 95(75-100)

G. cotidianus 31(24.6-37.0) 3.9 (NR) test not valid >100

NR = not reliable

4.8 Summary of standardised freshwater WETT protocols developed.

Standard protocols have been developed for a chronic growth inhibition test with the alga S. capricornutum (Appendix 4), and short term acute lethality tests for the invertebrates P. fluviatilis (Appendix 5) and C. dubia (Appendix 6) and the bully G. cotidianus (Appendix 7). The alga and the invertebrates all show sensitivity in the lower 20% of aquatic species tested for Zn sensitivity (USEPA 1987). Use of these standardised protocols for routine WETT is likely to provide a high level of protection for freshwater ecosystems. G. cotidianus however was only moderately sensitive to the reference toxicants used and use of O. mykiss as a test species may provide better protection for the native fish populations.

4.9 Further development of WETT protocols

It should be recognised that the protocols detailed in this report are working documents that need to be reviewed and updated on a regular basis. The application and use of these protocols with a variety of effluents will highlight areas that need to be reassessed and/or updated in future.


There is also a need for further evaluation of the sensitivity of native marine and freshwater fish species and the development of protocols for chronic WETT with native species.

5. Use of whole effluent toxicity tests

Whole effluent toxicity testing can be used in a variety of ways in the management of wastewater discharges. For example in

(i) Resource Consent conditions

(ii) Resource Consent applications

(iii) Toxicity Identification Evaluation (TIE).

Two key recommendations from the ‘Contaminant Toxicity in Aquatic Ecosystems: Tools and Approaches for New Zealand’ workshop held in Wellington in May 1998 in support of this project, were:

(i) That MfE take a co-ordinating role in the dissemination of information to regional councils and other interested parties regarding the ANZECC Water Quality Guidelines and the Whole Effluent Toxicity Testing protocols.

(ii) That MfE establish a working group to develop guidelines for the use of Whole Effluent Toxicity Testing in New Zealand.

The following section highlights some of the technical issues that will need to be addressed in these guidelines.

5.1 Use of WETT in Resource Consent Conditions

Although toxicity is not referred to, the use of WETT can provide a convenient means to assess the potential for an effluent to produce no ‘adverse effects’ or no ‘significant adverse effects’ on an ecosystem. The realisation that the use of chemical guidelines was not always providing the desired protection from toxic contaminant effects (Bergman et al. 1986) led a number of countries such as USA, Canada, Netherlands and Australia to develop WETT protocols for both acute and chronic tests. The failure of the chemical guidelines was primarily due to the fact that not all the


toxicants in a complex effluent and their interactions were being considered. In the USA in the late 1970s and early 1980s a series of acute WETT protocols were developed. These were followed by the development of chronic WETT protocols, guidelines and regulations associated with their use in the control of wastewater discharges USEPA 1991b, 1994, 1995a). Currently in the USA 6500 discharge permits have either WETT monitoring or compliance limits, or both (Heber et al. 1996). Canada has also incorporated extensive WETT into regulations for the pulp and paper industry (Environment Canada 1991).

In New Zealand WETT testing has been conducted since the mid 1980s, primarily with species that have been used overseas (Freeman 1986, Hickey 1995). In recent years, it has been acknowledged that WETT can be a useful tool in resource consent conditions, leading to the current development of protocols with native species.

5.1.1 Current use of WETT in Resource Consents in New Zealand

A recent review of resource consent conditions involving WETT protocols in New Zealand revealed fifteen consents with requirements for toxicity testing. Ten of these had conditions requiring monitoring and five had compliance conditions. The conditions were highly variable in both the approach and protocols used.

The following are examples of conditions requiring monitoring.

Example 1

Within six months of the date of commencement of this consent the consent holder, or its agent, shall carry out whole effluent toxicity tests on representative samples of its condensate, cooling water, cleaning water, and stormwater discharge to the river.

The whole effluent toxicity testing (WETT) should be carried out by a recognised practitioner using all three of:

• a freshwater species of algae

• a freshwater macroinvertebrate species

• a freshwater vertebrate species (juvenile fish or fish eggs)

An additional WETT may be requested annually at the peak of the dairy processing season.


Example 2

The grantee shall each day over a period of five consecutive days each year carry out daily effluent acute toxicity monitoring using a representative and sensitive test algal species. The grantee shall during one of the five days each year also carry out effluent acute toxicity monitoring using two representative and sensitive invertebrate species, preferably Deleatidium and Potamopyrgus if available and well proven as a test species. River water upstream of the wastewater treatment plant sewage outfall shall be used as the diluent in the above testing. This monitoring shall be undertaken when the contribution from the tannery is considered to be at a worst case. Both samples (effluent and diluent) shall be analysed for total Cu, Cr, Pb, Ni, Cd, Zn, phenols, chlorophenols and sulphide. The results shall be supplied to the Regional Council. This condition is subject to annual review.

Example 3

The toxicity of effluent shall be tested over a standard geometric dilution series (factor of 0.5) which encompasses the mixing ratio.

The mixing ratio shall be determined by the 95 percentile of the discharge flow rate for the previous twelve months divided by the mean annual 7-day low flow recorded immediately upstream of the discharge (16.6m3/s).

Toxicity testing shall be carried out using each of the following test protocols:

(a) Environment Canada (1990). Biological test method. Reference method for determining acute lethality of effluents to rainbow trout. Conservation and Protection, Ottawa, Ontario. EPS 1/RM/13.

(b) OECD (1981). Chronic reproduction test using a cladoceran (Daphnia magna). Test Method 202. In: Guidelines for the testing of chemicals. Organisation for Economic Co-operation and Development, Paris.

(c) Environment Canada (1992). Biological test method. Toxicity test using luminescent bacteria (Photobacterium phosphoreum). Conservation and Protection, Ottawa, Ontario. Report EPS 1/RM/24.

(d) Environment Canada (1992). Biological test method. Growth inhibition test using the freshwater alga Selenastrum capricornutum. Conservation and Protection, Ottawa, Ontario. Report EPS 1/RM/25.


The following two examples are compliance conditions which vary in the way in which toxicity is defined and potentially in the species used.

Example 4

The Consent Holder will notify the Council once the stormwater treatment ponds are full, and will initiate stormwater monitoring. The monitoring programme shall be as follows. Monitoring of the settling pond water will be carried out annually and monitoring of the storage pond every six months. On each monitoring occasion a single grab sample will be taken from near the pond outlets. Water samples will be tested for toxicity using three representative marine species. There shall be no significant toxicity after a 200 fold dilution. Significant toxicity is defined as a greater than 10% response over that of a control. The control used will be an uncontaminated sample of harbour water, collected on an incoming tide at the harbour entrance.

Example 5

Whole Effluent Toxicity, Metals, and Organic Compounds

(a) The testing for Whole Effluent Toxicity, Metals and Organic Compounds and shall be carried out on the samples.

(b) Whole Effluent Toxicity

(i) Tests shall use upstream river water as diluent.

(ii) The test organisms used in acute toxicity tests shall be:

Cladoceran Daphnia magna

Freshwater alga Selenastrum capricornutum

Bacterium Photobacterium phosphoreum (Microtox ™)

(c) “No toxicity” is defined as “The EC50 value for the most sensitive of three test organisms shall represent an in river dilution of no more than 15”


The examples of resource consent conditions given above highlight the variability in the level of detail specified for effluent sampling and WETT procedures. In terms of sampling the effluent, only some consents specify the type of sample to be taken i.e., a grab sample. Examples 2 and 4 define what water will be collected and used for the controls and dilution of the effluent. There is also variability in the detail of the species and protocols to be used. Example 1 specifies only the phylogenetic levels to be tested whereas Example 3 specifies both the species and protocols to be used. In terms of the dilutions of the effluent to be tested, Example 3 is very detailed and Examples 4 and 5, which are compliance conditions, specify dilutions in the definitions of what is considered toxic.

5.1.2 Recommendations for use of WETT in Resource Consent Conditions

The examples above highlight that the approaches to resource consent conditions throughout New Zealand are variable. It is recognised that each resource consent has different requirements, however this does not exclude the use of a consistent approach to the use of WETT in consent conditions. There are several key issues that need to be considered in the application of WETT to resource consent conditions.

(i) Use of standardised protocols

When monitoring or compliance conditions are set on a resource consent the species to be used should be identified. These species need to have standardised protocols and preferably be native species. The use of native species removes the question of relevance of non native species and the use of exotic species in regard to the Biosecurity Act (1993, Amended 1997). The protocols detailed in this report are obvious candidates for use in resource consent conditions. In some cases, for example where there is a high perceived environmental risk, it may be appropriate to use site specific species and protocols that have been developed for similar species overseas. For example the use of the standard D. magna (USEPA 1993b) protocol with the New Zealand daphnia species D. carinata (Hickey and Vickers 1994).

(ii) Range of species used

The species specified for use on the resource consent should ideally cover three phylogenetic levels, algae, invertebrates and fish to provide maximum protection for the receiving water ecosystem. This approach is widely recommended overseas (USEPA 1991b).

(iii) A definition of “no adverse effect” and “no significant adverse effect”.


The Resource Management Act (1991) focuses on the effects of a discharge at the level of the receiving water ecosystem. The Act provides that a regional council shall not grant a discharge permit if, after reasonable mixing, the discharge is likely to give rise to a “significant adverse effect” (s.107). Where waters are classified for aquatic ecosystem purposes (AE) under the Third Schedule, the Resource Management Act is more stringent and states that a discharge shall not be allowed if, after reasonable mixing, there is any “adverse effect” on aquatic life.

The definitions of “significant adverse effect” and “no adverse effect” are difficult and complex.

In the examples of current consent conditions given above, rather different approaches were taken. In Example 4 ‘no significant toxicity’ is defined as a greater than 10% response over that of the control. Example 5 ‘no toxicity’ provides a site specific definition of ‘no toxicity as “the EC50 value for the most sensitive of three test organisms shall represent an in river dilution of no more than 15”. This condition involved an acute EC50, an application factor of 0.05 (ANZECC 1992) and a site specific dilution factor of 300, to safeguard against chronic toxic effects after reasonable mixing.

Overseas there have been several different approaches to defining “no adverse effect” or “safe concentration”. In the USA for the determination of acceptable concentrations of toxic wastes, the USEPA has adopted “concentration criteria” which are based on a certain proportion of the effluent. This is basically the same as applying an application factor to toxicity data to derive an acceptable discharge concentration, although different terminology is used. Their calculation involves converting the measured toxicity (expressed in terms of an acute LC50 or EC50 or chronic NOEC to a toxicity unit (TU).

TULC or NOEC

=100

50

The “safe concentration” or criterion for the receiving water is then defined as 1.0 TU for chronic tests or 0.3 TU for acute tests. A toxic unit is specific to a particular test methodology and effluent. Two expressions for the allowable safe concentration are recommended: a criterion maximum concentration (CMC) to protect against acute (short term) effects, and a criterion continuous concentration (CCC) to protect against chronic (long term) effects. For acute protection, the CMC should not exceed 0.3 of the acute toxic unit, as measured by the most sensitive of at least three test species. For chronic protection, the CCC should not exceed the chronic toxic unit of the most sensitive of at least


three test species. The USEPA has found that application of 0.3 of the toxic unit for the CMC has the effect of adjusting an LC50 value to an LC1 (virtually no mortality). The factor 0.3 was derived from a comparison of data from 496 separate effluent toxicity tests on industrial and municipal effluents by the USEPA.

In Canada (Environment Canada 1996), Australia (ANZECC 1992) and in some USA States an arbitrary application factor is used to derive “safe concentrations” from the results of acute toxicity values. For toxicants that are considered to be nonpersistent in wastes the factor used is the acute toxicity LC50 value for the most sensitive species tested multiplied by 0.05 and for persistent wastes the factor is 0.01. Either the Toxicity Unit approach or the application factor approach could be used in New Zealand.

The River Ecosystems Management Framework currently being developed by a Ministry for the Environment working group is taking an ecotyping approach to assist in the development of site specific objectives for ecological values. This framework will then be used to develop site specific definitions for “no significant adverse effects” and “no adverse effects”.

When development of the River Ecosystem Management Framework reaches a more advanced stage, consideration needs to be given to how WETT can be integrated into this approach.

(iv) Type of test performed

Examples 4 and 5 require a different approach to the toxicity testing. In Example 4 the toxicity tests would be conducted using a hypothesis testing approach, where a simple comparison of the toxic response is made between a 200 fold dilution of the sample effluent and a control. The toxic response measured in the two samples would be compared using a standard statistical test. The ability of the hypothesis testing approach to discriminate toxicity differences between samples is very dependent upon the number of organisms and replicates used in the test. Also a single dilution test provides no confidence that the observed effect is actually caused by the toxicity. By comparing only two samples there is no assurance for example that the observed difference was not a result of some unrelated factor, such as disease in the test organisms, or accidental contamination of the sample, which are influences that would be identified by a concentration response approach. The hypothesis test also gives no indication of how far away from the toxic effect the regulatory concentration may be. The approach taken in Example 5 uses a test that involves a dilution series of the effluent. This then enables a 50% or other response levels to be robustly


identified using a dose response curve calculation. When a dilution series is used there should be a graded response observed at the various concentrations. The dilution series method establishes that there is a concentration response gradient over the dilution series which is a diagnostic test of toxicity. The concentration response is a more robust method for predicting low threshold effects and is recommended for routine use in resource consent conditions.

(v) Use of mixing zones

Example 3 raises the issue of mixing zones and how they should be used in a resource consent condition. There are two key issues associated with mixing zones, the definition of the mixing zone and also the issue of effects acceptable within a mixing zone. The consideration of mixing zones is a key issue in the RMA (1991) as there is a requirement (s.107(1)(g)) that the consent cannot be granted if, after a ‘reasonable mixing’ there is likely to be ‘any significant adverse effect’ or in the case of Class AE waters the stricter requirement of ‘no adverse effect on aquatic life’ must be considered. In a discussion document on ‘reasonable mixing’ Rutherford et al. (1994) suggested that reasonable mixing may be said to have occurred when the management objectives of the receiving water are not compromised by the non-compliance zone. This will require an assessment of the size and location of the non-compliance zone, and the conditions within it. They suggested that:

• Generally, the size of the non-compliance zone should be minimised,

• Any adverse effects should be confined to the non-compliance zone, and

• Any adverse effects within the non-compliance zone should be minor.

The River Ecosystem Management Framework working group is also addressing the issue of mixing zones and will be making recommendations for a site specific approach to be taken.

A rather different approach is currently being used in the USA where the WETT Control Policy (USEPA 1994) recommends evaluation of WETT for both acute and chronic toxicity at:

• end of pipe

• mixing zone edge for acute toxicity

• mixing zone edge for chronic toxicity


Requirements vary between discharge permits and one requirement is for no or low toxicity at the end of the pipe. In another part of the mixing zone there can be no acute toxicity, and in another part, no chronic toxicity. If there is to be no acute toxicity 10m from the outfall, the dilution at 10m is measured and appropriate dilutions tested using a toxicity test. These results are then compared the acute NOEC for the required mixing zone dilution.

In the future the USEPA is looking to eliminate the concept of mixing zones by 2004 for Bioaccumulative Chemicals of Concern (BCC’s). However, Smith & Carr (1993) note that elimination of mixing zones to protect wildlife or human health from BCC’s has little scientific merit. The principle assumes that the population of interest resides at the end of the pipe.

British Columbia (Canada) does not allow a mixing zone for certain chemicals in discharges.

Guidance needed for general application is determined by:

(a) sensitivity of the receiving environment in terms of species present and available dilution;

(b) specific consideration regarding the speed-of-action of the toxicant and potential for effects in the mixing zone (e.g., some pesticides are very rapid acting).

(vi) Variability in WETT results

The potential use of WETT as a compliance condition in resource consents means that test variability must be well understood so that appropriate conditions can be set. In 1995 the Annual Pellston Workshop focused on evaluation of the strengths and weaknesses of the USEPA WETT programme to identify areas for improvement. One of the sessions at this workshop focused on the variability in WETT. The variability in WETT arises from a wide range of factors with the key to variability being characteristics of test conditions and associated experimental design factors. Burton et al. (1996) identified a series of factors that can influence test results (Table 15) and a set of factors that can reduce test variability (Table 16).


Table 15. WETT test factors that can influence test result variability

Test factors Effect on variability

Sampling procedures Considerable

Sample representativeness Considerable

Frequency of subsampling of composite Considerable

Sample holding time Considerable

Deviation of temperature Considerable

Deviation of test duration Considerable

Deviation from required light Slight (except algae)

Deviation from required feeding Considerable

Dilution water Considerable

Deviation from control performance criteria Moderate

Table 16. WETT test factors that can reduce test result variability

Test factors Effect on variability

Increase analyst experience Greatly decreases

Improve analyst technique Greatly decreases

Improve organism quality Greatly decreases

Increase minimum replicates Moderately decreases

Increase minimum concentrations Moderately decreases

Increase minimum number of organisms Slightly decreases

The two key factors identified as being of most concern were:

(i) analyst experience, this affects proper implementation of test procedures and interpretation of data generated

(ii) test organism condition/health

These factors should be considered when New Zealand guidelines are being developed as should several of the recommendations of the workshop which are relevant to the New Zealand situation. These recommendations were;

(i) establish WETT test specific variability limits

(ii) develop and implement quality assurance and audit programmes


(iii) develop guidance on data interpretation regarding WETT variability.

There is the opportunity to learn from experience elsewhere when developing the initial guidelines for New Zealand.

5.2 Use of WETT in Toxicity Identification Evaluations (TIE)

Linked to the use of WETT on discharge consents in some countries is the use of WETT methodology in TIE procedures. The TIE procedures are used in the USA and Canada when unacceptably toxic effluents have been identified and in some cases dischargers are required to perform Toxicity Reduction Evaluations (TRE’s) as a condition of their discharge permits. A TRE is defined as:

“a site specific study conducted in a stepwise process designed to identify the causative agents of effluent toxicity, isolate the sources of toxicity, evaluate the effectiveness of toxicity control options, and then confirm the reduction in effluent toxicity: Toxicity identification evaluations (TIE’s), which are a part of the TRE, consist of methods to characterise and confirm the cause of acute and chronic toxicity in effluents. TIE’s use standard acute and chronic WETT protocols (USEPA 1993, 1995b) to detect the presence of toxicants in the effluent. The aim is to identify the key toxic components of the effluent. To achieve this the number of constituents in the effluent is reduced by moving up the effluent pipe. The effluent is then manipulated either chemically or physically to remove certain types of compounds (i.e., oxidants, cationic metals, volatiles, non-polar organics). These manipulations may involve for example:

(i) addition of EDTA to produce non-toxic complexes with cationic metals

(ii) addition of sodium thiosulphate to reduce the toxicity of oxidative compounds such as iodine, bromine, ozone, chlorine

(iii) filtration of the effluent to identify toxicity associated with suspended solids or removable particle-bound toxicants.

(iv) alteration of pH to identify substances which have pH dependent toxicity such as hydrogen sulphide and ammonia.

By using a series of manipulations (as outlined in USEPA 1992) the source of the toxicity in a complex effluent can often be identified. This knowledge can then be used to reduce the toxicity of the effluent.


TIE has rarely been used in New Zealand to date. It is however a useful tool for both resource managers and dischargers and inclusion of guidelines for TIE procedures should be considered in the overall guideline for use of WETT in New Zealand.

5.3 Use of WETT in Resource Consent Applications

The use of a tiered integrated investigative approach involving chemical, WETT and biological monitoring (Hickey & Roper 1992, Martin et al. 1998) has been suggested for impact assessments. The three tier approach using WETT in each tier was recommended for the assessment of effluents in New Zealand. This was based on approaches developed by the USEPA (1989, 1991b).

The first tier would include a basic screening of the effluent for acute toxicity, the second tier a set of chronic toxicity tests and the third tier a site specific approach. The factors which were suggested for consideration in determining the level of the investigation were:

• environmental risk

• dilution factor available for effluent

• chemical composition of effluent

• public pressure

• results of previous tests

The suggested approach recommends that the initial screening tier involves standardised acute WETT primarily with New Zealand native species on a single sample.

The second tier would involve WETT with at least three native species from three phylogenetic levels to be carried out on more than one sample. If no acute toxicity occurs, chronic WETT with native species would be recommended on multiple samples. This tier may also include some site specific testing of relevant species using a basic standard protocol for a similar overseas species. Chemical analyses of the samples would also be recommended.

The third tier would involve all of the analyses conducted in tier 2 on multiple samples, as well as biological monitoring of the receiving waters and benthic communities. Use of site specific species tests would be advisable. There are a large number of these that have been used in New Zealand. These include the use of D. carinata, Deleatidium sp. (Hickey and Vickers 1994) and also the use of seasonal species like whitebait.


6. References

Anderson, B.S. et al. (1990). Procedures manual for conducting toxicity tests developed by the marine bioassay project. Vol 90-1-WQ. Water Resources Control Board, State of California.

ANZECC (1992). Australian Water Quality Guidelines for Fresh and Marine Waters. Australian and New Zealand Environmental and Conservation Council.

ASTM (1997). Biological Effects and Environmental Fate; Biotechnology, Pesticides. Annual Book of ASTM Standards 11.05.

Beckman. (1982). Microtox operating system manual. Beckman Instruments Inc.

Bergman, H.L.; Kimerle, R.A.; Mabi, A.W. (1986). Environmental hazard assessment of effluents. Elmsford NY: Pergamon.

Burton, A. et al. (1996). Effluent Toxicity Test Variability. In Whole Effluent Toxicity Testing: an evaluation of methods and prediction of receiving system impacts. Ed. Grothe, D.R.; Dickson, K.L.; Reed-Judkins, D.K. SETAC Press, Pensacola.

Environment Canada (1990a). Biological test method: Reference method for determining acute lethality of effluents to rainbow trout. EPS 1/RM/13.

Environment Canada. (1990b). Biological test method: acute lethality test using rainbow trout + amendment. EPS 1/RM/9. Environment Canada.

Environment Canada. (1990c). Biological test method: acute lethality test using Daphnia spp. EPS 1/RM/11.

Environment Canada. (1990d). Biological test method: acute lethality test using threespine stickleback (Gasterosteus aculeatus). EPS 1/RM/10.

Environment Canada. (1990e). Guidance documentation. Control of toxicity test precision using reference toxicants. EPS 1/RM/12.

Environment Canada (1991). Pulp and paper effluent regulations. Public Information Version. Ottawa, Ontario. Environmental Protection, Conservation and Protection. January 1991.


Environment Canada. (1992a). Biological test method: growth inhibition test using the freshwater alga Selenastrum capricornutum, EPS 1/RM/25. Environment Canada.

Environment Canada. (1992b). Biological test method: toxicity test using luminescent bacteria (Photobacterium phosphoreum). EPS 1/RM/24.

Environment Canada. (1992c). Biological test method: toxicity tests using early life stages of salmonid fish (Rainbow Trout, Coho Salmon, or Atlantic Salmon). EPS 1/RM/28.

Environmental Canada (1992d). Biological test method: Fertilisation Assay Using Echinoids (Sea urchins and Sand dollars). EPS 1/RM/27.

Environment Canada (1997a). Biological Test Method: Test for survival and growth in sediment using the freshwater amphipod Hyalella azteca. EPS 1/RM/33.

Environment Canada (1997b). Biological Test Method: Test to reproduction and survival using cladoceran Ceriodaphnia dubia. Amendments to EPS 1/RM/21.

Environment Canada (1997c). Biological test method: Test of larval growth and survival using fathead minnows. Amendments to EPS 1/RM/22.

Environment Canada (1997d). Biological test method: Fertilization assay using echinoids (sea urchins and sand dollars). Amendments EPS 1/RM/27.

Environment Canada 1997e. Biological test method: Growth inhibition test using freshwater alga Selenastrum capricornutum. Amendments to EPS 1/RM/25.

Freeman, M.C. (1986). Aquatic toxicity tests: Comparative assessment of four acute tests and their potential application to New Zealand. Ministry of Works and Development, Wellington. p. 36.

Gunthorpe, L.; Nottage, M.; Palmer, D.; Wu, R. (1997a). Testing for sublethal toxicity using gametes of Hormosira banksii. National Pulp Mills Research Programme. Technical Report No. 22.

Gunthorpe, L.; Nottage, M.; Palmer, D.; Wu, R. (1997b). Development and validation of a fertilisation inhibition bioassay that uses gametes of Hormosira banksii. National Pulp Mills Research Programme. Technical Report No. 21.


Heber, M.A.; Reed-Judkins, D.K.; Davies, T.T. (1996). USEPA’s Whole Effluent Toxicity Testing Program: a national regulatory perspective. In: Whole Effluent Toxicity Testing: an evaluation of methods and prediction of receiving system impacts. Ed. Grothe, D.R.; Dickson, K.L.; Reed-Judkins, D.K. SETAC Press, Pensacola.

Hickey, C.W. (1989). Sensitivity of four New Zealand cladoceran species and Daphnia magna to aquatic toxicants. New Zealand Journal of Marine and Freshwater Research, 23: pp. 131-137.

Hickey, C.W.; Roper, D.S. (1992). A review of toxicity testing methods for marine waters. Auckland Regional Council, Environmental and Planning Division. Hamilton. September.

Hickey, C.W.; Vickers, M.L. (1994). Toxicity of ammonia to nine native New Zealand freshwater invertebrate species. Arch. Environ. Contam. Toxicol. 26: pp. 292-298.

Hickey, C.W. (1995). Ecotoxicity in New Zealand. Journal of Australasian Ecotoxicity 1: pp. 43-50.

Hooten, R.L.; Carr, R.S. (1998). Development and application of a marine sediment pore-water toxicity test using Ulva facciata zoospores. Environm. Toxicol. Chem. 17: pp. 932-940.

Martin, M.L.; Hickey, C.W.; Hall, J. (1998). Assessment of environmental effects using toxicity testing. Water and Wastes in New Zealand, March. pp. 35-57.

Pyle, E. (1996). A proposed methodology for deriving aquatic guideline values for toxic contaminants: document for public comment. Pollution and Risk Management Directorate, MfE. June 1996.

Quinn, J.M.; Hickey, C.W. (1990). Characterisation and classification of benthic invertebrate communities in 88 New Zealand rivers in relation to environmental factors. NZ Journal of Marine and Freshwater Research 24: pp. 387-409.

Rand, G.M.; Petrocelli, S.R. (1985). Fundamentals of aquatic toxicology. 665p. Hemisphere Publishing, New York.

Rutherford, K.; Zuur, R.; Race, P. (1994). Resource Management Ideas. No. 10. A discussion on reasonable mixing in water quality management.


Schimmel, S.C.; Morrison, G.E.; Heber, M.A. (1989). Marine complex effluent toxicity program: test sensitivity, repeatability and relevance to receiving water toxicity. Environmental Toxicology and Chemistry 8: pp. 739-46.

Smith, D.; Carr, B. (1993). Designing goals for the Great Lakes. Water Environment and Technology. June: pp. 47-51.

USEPA (1987). Ambient water quality criteria for Zn 1987. EPA 440/5-87-003.

USEPA. (1989). Biomonitoring for control of toxicity in effluent discharges to the marine environment. Vol. EPA/625/8-89/015. US Environmental Protection Agency, Centre for Environmental Research Information, Office of Research and Development, Cincinnatti, OH 45268.

USEPA. (1991a). Policy on the use of biological assessments and criteria in the water quality program. Washington DC: USEPA Office of Water.

USEPA. (1991b). Technical support document for water quality-based toxics control. EPA-505/2-90/001. US Environmental Protection Agency.

USEPA. (1992). Interim Guidance on Interpretation and Implementation of Aquatic Life Criteria for Metals. USEPA, Washington.

USEPA (1993). Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms. EPA/600/4-90/027F. August 1993.

USEPA. (1994). Whole effluent toxicity (WET) control policy. Washington DC: USEPA Office of Water. EPA/833-B-94-002.

USEPA. (1995a). National policy regarding whole effluent toxicity enforcement. Washington DC: USEPA Offices of Regulatory Enforcement and Wastewater Management.

USEPA (1995b). Short-term methods for estimating the chronic toxicity of effluents and receiving waters to West Coast Marine and Estuarine Organisms. EPA/600/R-95/136.

Walsh, G.E. (1987). Methods for toxicity tests of single substances and liquid complex wastes with marine unicellular algae. EPA-600-8/87/043. Cincinnati, Ohio. US Environmental Protection Agency.


7. Terminology

Note: All definitions are given in the context of this report, and may not be appropriate in another context.

Chemical is, in this report, any element, compound, formulation, or mixture of a chemical substance that may enter the aquatic environment through spillage, application, or discharge. Examples of chemicals which are applied to the environment are insecticides, herbicides, fungicides, sea lamprey larvicides, and agents for treating oil spills.

Control is a treatment in an investigation or study that duplicates all the conditions and factors that might affect the results of the investigation, except the specific condition that is being studied. In an aquatic toxicity test, the control must duplicate all the conditions of the exposure treatment(s), but must contain no test material. The control is used to determine the absence of measurable toxicity due to basic test conditions (e.g., quality of the control/dilution water, health or handling of test organisms).

Control/Dilution water is the uncontaminated water used for the sample control and for dilution of the test substance to prepare different concentrations for the various treatments. It may be uncontaminated receiving water or previously collected oceanic water prior to discharge.

Culture as a noun, means the stock of organisms raised under defined and controlled condition to produce healthy test organisms. As a verb, it means to carry out the procedure of raising organisms.

Effluent is any liquid waste (e.g., industrial, municipal) discharged to the aquatic environment.

Monitoring is the routine (e.g., daily, weekly, monthly, quarterly) checking of quality or collection and reporting of information. In the context of this report, it means either the periodic (routine) checking and measurement of certain biological or water-quality variables, or the collection and testing of samples of effluent, elutriate, leachate, or receiving water for toxicity.


Receiving water is surface water (e.g., in a stream, river, or lake) that has received a discharged waste, or is about to receive such a waste (e.g., it is just upstream from the discharge point). Further descriptors must be provided to indicate which meaning is intended.

Reference toxicant is a standard chemical used to measure the sensitivity of the test organism in order to establish confidence in the toxicity data obtained for a test material. In most instances a toxicity test with a reference toxicant is performed to assess both the sensitivity of the organisms at the time the test material is evaluated, and the precision of results obtained by the laboratory.

Wastewater is a general term which includes effluents, leachates, and elutriates.

Whole effluent is any liquid waste (e.g., industrial, municipal) discharged to the aquatic environment.

Toxicity Terms

Acute toxicity is a discernible adverse effect (lethal or sublethal) induced in the test organisms within a short period of exposure to a test material relative to the life span of the organism.

Chronic toxicity implies long-term effects that are related to changes in metabolism, growth, reproduction, or ability to survive. In this test, chronic toxicity is a discernible adverse effect (lethal or sublethal) induced in the test organism during a significant and sensitive part of the life-cycle.

EC50 is the effective concentration (i.e., the concentration of material in water that is estimated to produce a specifically quantified effect to 50% of the test organisms). The EC50 and its 95% confidence limits are usually derived by statistical analysis of a quantal, “all or nothing”, response (such as death, fertilization, germination, or development) in several test concentrations, after a fixed period of exposure. The duration of exposure must be specified (e.g., 72 h EC50).


End point means the variables (i.e., time, reaction of the organisms, etc.) that indicate the termination of a test, and also means the measurement(s) or value(s) derived, that characterise the results of the test (LC50, LT50, etc.)

LOEC lowest observed effect concentration. The lowest concentration tested causing a statistically measurable effect to the test system

NOEC no observed effect concentration. The highest concentration tested causing no statistically measurable effect to the test system.

Toxicity is the inherent potential or capacity of a material to cause adverse effects on the test organism.

Toxicity test is a method to determine the effect of a material on a group of selected organisms under defined conditions. An aquatic toxicity test usually measures either (a) the proportions of organisms affected (quantal) as measured by EC50, or (b) the degree of effect shown (graded or quantitative) after exposure to specific concentrations of whole effluents or receiving water as measured by an EC50.

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