5. Testing strategies
5.2 Refinement of PNEC: strategy for further testing
5.2.2 Aquatic compartment
5.2.2.2 Available long-term tests
The long-term tests available when seeking to refine the effects assessment are limited, but it is nevertheless important that the correct test is chosen to maximise the usable information and avoid unnecessary repeat testing.
Long-term fish testing
Fish early-life stage (FELS) test (OECD 210)
This test is considered as the most sensitive of the fish tests, covering several life stages of the fish from the newly fertilised egg, through hatch to early stages of growth. This is felt to cover most, but not all, of the sensitive points in the life-cycle, and is the only suitable test currently available for examining the potential toxic effects of bioaccumulation, apart from the full life cycle test. It is, however, a long test, typically 60 days post-hatch for rainbow trout (Oncorhynchus mykiss), or approximately 30 days post-hatch for warm water fish, and is consequently the most expensive of those available. It should be requested where long-term fish toxicity data are required and the substance has the potential to bioaccumulate.
Egg and sac-fry stage test (draft guideline)2
This test measures the sensitive early life stages from the newly fertilised egg to the end of the sac-fry stage. It is considerably shorter, and hence cheaper, than the FELS test but is also considered to be less sensitive. The method is currently the subject of discussion at OECD, and is available as a draft method. It offers an alternative to the FELS test for substances with log Kow less than 4. The conditions under which the egg and sac-fry stage test can be used in place of the FELS test may be clarified following the further discussion at the OECD.
28-day growth test (draft guideline)2
This test measures the growth of juvenile fish over a fixed period, and is considered a sensitive indicator of fish toxicity. Although it is considered to be of insufficient duration to examine all the sensitive points in the fish life-cycle, it provides a shorter and cheaper option to the FELS test for substances of log Kow < 5. It is currently the subject of discussion at OECD and is available as a draft method.
Fish, prolonged toxicity test, 14-day study (OECD 204)
This test cannot be considered as a suitable long-term toxicity study since it does not examine a sensitive stage in the fish life-cycle. It is, in effect, a prolonged acute study with fish mortality as the major end-point examined. However, sub-lethal effects are monitored and the NOEC should be based on the absence of these effects. It should not be requested where a long-term fish study is required. It should only be requested where further information on possible short-term effects is considered necessary.
Long-term Daphnia testing
14-day Daphnia reproduction test (OECD 202, part II)
This test measures the juvenile production as well as parental immobility and mortality. It is frequently (and preferably) conducted over 21 days. Although it does not cover the full life cycle of Daphnia it covers the sensitive reproduction stage. It is therefore considered a sensitive long-term study. It has, however, generated a number of problems, including reproducibility, and is being revised in a new guideline.
21-day Daphnia reproduction test (revised OECD 202, part II, draft)
This test is a development of the above procedure with a number of important modifications to improve the reliability and reproducibility. It is likely to be adopted as a replacement guideline by OECD after the completion of a ring test.
Algal testing:
Algae toxicity test (EEC C3)
The algal growth inhibition test measures the inhibition of algal growth under standard conditions of light, temperature and nutrient concentrations. The test produces an EC50, which can be considered as equivalent to a short-term L(E)C50. The lowest of ErC50 and EbC50 (i.e. the EC50 measured according to the growth rate and biomass respectively, Nyholm, 1985, 1989) should be used. It is not only a multigeneration test but also measures the sublethal effect, reduction in population growth, and therefore can be considered as a true chronic test. The NOEC may therefore be used in the testing strategy.
5.2.2.3 Decision table for further testing
The decisions to be made on further testing are detailed in Table 18 and take the basic criteria above into account, although common sense must be applied to such generalised rules when considering individual situations. The decisions taken on further testing will be different depending on species sensitivity. In all cases, the algal study from the base set is first considered as a short-term study and the EC50 used for calculation of the PNECwater. However, the algal study is technically a multi-generation test and thus, if there are other long-term NOEC data, the algal NOEC can be considered as a long-term NOEC in the revised assessment. Generally, this algal NOEC would not be used unsupported by other long-term data.
Chapter 4 (Use of (Q)SARs) gives full details on the use of the QSAR estimates for substances with a non-specific mode of action and on long-term fish and Daphnia toxicity within the testing strategy.
Table 18 Decision table for aquatic toxicity testing when results from a full base-set (FBS(a)) using an assessment factor on the lowest L(E)C50, show that PEC/PNEC>1
Variation in base-set data
Further testing Data available for assessment
Assessment factor(b) No significant
diffe-rence between the L(E)C50 values of fish, Daphnia or algae
A1 Long-term fish test + long-term Daphnia test + determination of NOEC algae
FBS + algae +
Daphnia + fish 10
Fish LC50 more than 10 times lower than L(E)C50 of Daphnia and algae
A2 Long-term fish test + determination of NOEC algae
If S/L(c) ratio for fish > 20:
long-term Daphnia test(d)
FBS + algae + fish
FBS + algae + fish + Daphnia
50
10 Daphnia L(E)C50
more than 10 times lower than L(E)C50
of fish and algae
A3 Long-term Daphnia test + deter-mination of NOEC algae If S/L(c) ratio for Daphnia >
20: long-term fish test(d)
FBS + algae + Daphnia
FBS + algae + fish + Daphnia
50
10 Algae L(E)C50 more
than 10 times lower than L(E)C50 of fish and Daphnia
A4 Test on other algae species + long-term fish/Daphnia test(e)
FBS + two algae(e)
+ fish/Daphnia 10(e)
Fish LC50 more than 10 times higher than L(E)C50 of Daphnia and algae
A5 Long-term Daphnia test + determi-nation of NOEC algae
If S/L(c) ratio for Daphnia >20;
long-term fish test(d)
FBS + algae + Daphnia
FBS + algae + fish + Daphnia
50
10 Daphnia L(E)C50
more than 10 times higher than L(E)C50
of fish and algae
A6 Long-term fish test + determinati-on of NOEC algae
If S/L(c) ratio for fish >20: long-term Daphnia test(d)
FBS + algae + fish
FBS + algae + fish + Daphnia
50
10 Algae L(E)C50 more
than 10 times higher than L(E)C50 of fish and Daphnia
A7 Long-term Daphnia test + long-term fish test + delong-termination of NOEC algae
FBS + algae + fish +
Daphnia 10
NOTES:
(a) FBS = full base set which includes L(E)C50 values for fish, Daphnia and algae.
(b) AF = the assessment factor must be applied to the lowest NOEC available at this stage, including the NOEC from the algae test.
(c) S/L refers to the short-term to long-term ratio, i.e. the ratio between the L(E)C50 from a short-term test and the NOEC from a long-term test.
(d) Generally testing of a third species will be unnecessary since the toxicity results from the first species should be protective. However, this cannot be a fixed rule given the toxicity variations within taxonomic groups as well as between them. Thus if a short-term L(E)C50: long-term NOEC ratio > 20 is found for the species tested, or from the algal study, then further testing of a third species might be necessary. The use of long-term fish or Daphnia QSARs could help in deciding which species need to be tested (see Chapter 4 "Use of QSARs"). It is considered that such a ratio may be indicative of an abnormal level of toxicity or a specific mode of action, and thus the acquisition of additional evidence is justified in order to improve the confidence in the calculated PNECwater. Other factors such as the shape of the toxicity time curve and the presence of sub-lethal effects in the short-term toxicity study for the second species may also be considered. An assessment factor of 10 may be applied to the lowest of the three NOECs. Before a toxicity study on a third species is requested, due consideration should be given to whether the resultant NOEC will lead to a further revision of the PNECwater.
(e) This table is based on the presumption that an algal NOEC is available at the base-set.
If this is not the case an assessment factor of 50 should be used .
5.2.3 Soil compartment
At the moment there is a lack of standardised ecotoxicity tests for terrestrial organisms. The OECD tests on plants and earthworms are the only ones which are directly related to the soil compartment. These tests are of relatively short duration and should be regarded as short-term tests.
Several research programmes have been started in the last years, aimed at the development of soil tests: the Netherlands Integrated Soil Research Programme (NISRP; Eijsackers, 1989) and the Swedish Mark Test System (MATS; Rundgren et al., 1989). Recently, in Denmark a research programme has been started in which attention is paid to terrestrial toxicity research.
Finally, within the framework of the EU Environment Programme the international research project Sublethal Effects of Chemicals on Fauna Soil Ecosystem (SECOFASE) receives funding for the development, improvement and standardisation of ecotoxicity tests with a number of terrestrial organisms (Løkke and Van Gestel, 1993). However, it is unclear at the moment how many and which ecotoxicity tests should be implemented in testing strategies for a realistic effect assessment for the soil compartment. This means that a set of tests according to the base-set for the water compartment (algae, Daphnia and fish) does not exist for the soil compartment. A test set for soil organisms could be devised to contain data on:
• primary producers (plants);
• consumers (herbivores, fungivores and saprovorous organisms);
• decomposers (litter consumers and micro-organisms).
Within the OECD Test Guidelines Programme a suitable test set for soil organisms is being developed. Within this framework Léon and Van Gestel (1994) made an inventory of existing and international test guidelines and newly developed tests. Also they made an inventory of criteria for the selection of toxicity tests, and developed a more or less standardised method for selecting tests based on a scoring system for these criteria.
In the "OECD Terrestrial Effects Working Group Meeting" in June 1995 types of terrestrial testing required for various chemicals were discussed. For "general" chemicals the following tests were recommended at an "initial stage" (OECD, 1995):
• plant test involving exposure via soil;
• test with an annelid (earthworm acute or possibly reproduction test);
and/or
• test with a soil-dwelling arthropod.
Several tests with soil organisms are summarised in Appendix VI.
With respect to the testing strategy two cases can be distinguished if it is decided to revise the PNECsoil:
(1) If the equilibrium partitioning method is applied due to the absence of toxicity data for soil organisms and PECsoil/PNECsoil is >1, short term tests on primary producers, consumers and decomposers should be performed. Test guidelines are available for primary producers (OECD 208) and for consumers (OECD 207). Tests for decomposers have been developed for plant protecting agents and are available in several EU Member States as tests standardised on a national level (e.g. NEN (1988) in the Netherlands, BBA (1990b) in Germany). Currently, more methods are being developed (see Appendix VI).
(2) If the PNECsoil is based on toxicity data for soil organisms using assessment factors and PECsoil/PNECsoil >1, further testing may be necessary.
Depending on the effect a substance has on vascular plants, earthworms or micro-organisms, the information about the ecotoxicological effect on the most sensitive group of organisms (primary producers, consumers or decomposers) has to be improved by conducting appropriate tests for the respective endpoints. The choice of test species will be made on a case-by-case basis taking into account the availability of a suitable test method, the indicative nature of the assessment factors and the uncertainty in the proposed approach.
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