Table 6.
Validity criteria in OECD aquatic studies and their relevance for TKTD
| Guideline | Validity criteria in the unmodified test guideline (plus some additional requirements) | Relevance of these criteria for data underlying TKTD modelling and where changes are needed |
|---|---|---|
| Fish acute toxicity test (OECD, 1992) | The mortality in the control(s) should not exceed 10% (or one fish if less than 10 are used) at the end of the test |
This criterion is directly relevant as it demonstrates suitable experimental conditions for the fish This is also relevant for calibration of background mortality |
| Constant conditions should be maintained as far as possible throughout the test and, if necessary, semi‐static or flow‐through procedures should be used (see Annex 1 of the guideline for definitions) | This criterion is relevant for all conditions except where variation is intentional (i.e. to generate pulses of exposure) | |
| The dissolved oxygen concentration must have been at least 60% of the air saturation value throughout the test | This criterion is directly relevant as it relates to ensuring suitable conditions | |
| There must be evidence that the concentration of the substance being tested has been satisfactorily maintained, and preferably it should be at least 80% of the nominal concentration throughout the test. If the deviation from the nominal concentration is greater than 20%, results should be based on the measured concentration |
Concentrations over time should be measured so it is clear what the fish were exposed to. Instead of using a mean measured concentration the whole exposure profile is relevant (whether the study aims at constant or time‐variable exposure) Maintaining constant exposure is not required to use the study for TKTD but the exposure profile should be described. This applies even if the study aimed for constant exposure but did not achieve it |
|
| Fish early life stage toxicity test (OECD, 2013) | The dissolved oxygen concentration should be > 60% of the air saturation value throughout the test | This criterion is directly relevant as it relates to ensuring suitable conditions |
| The water temperature should not differ by more than + 1.5°C between test chambers or between successive days at any time during the test, and should be within the temperature ranges specified for the test species (Annex 2 of the guideline) |
This criterion is directly relevant as it relates to ensuring suitable conditions If a physiological model that includes variable temperature is used this could also be applied to the study |
|
| The analytical measure of the test concentrations is compulsory | This is particularly important for TKTD modelling where the whole exposure profile is relevant | |
| Overall survival of fertilised eggs and post‐hatch success in the controls and, where relevant, in the solvent controls should be greater than or equal to the limits defined in Annex 2 of the guideline. | This criterion is directly relevant as it demonstrates suitable conditions for the fish | |
| Daphnia sp. acute toxicity test (OECD, (2004a) | In the control, including the control containing the solubilising agent, not more than 10% of the daphnids should have been immobilised |
This criterion is directly relevant as it demonstrates suitable conditions for the daphnids This is also relevant for calibration of background mortality |
| The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels | This criterion is directly relevant as it relates to ensuring suitable conditions | |
|
ALSO (not a validity criteria): The concentration of the test substance should be measured, as a minimum, at the highest and lowest test concentration, at the beginning and end of the test |
It is important that the test concentrations are measured, but the minimum requirements are likely to be greater for TKTD modelling as information is required on the whole exposure profile Maintaining constant exposure is not required to use the study for TKTD but the exposure profile should be described. This applies even if the study aimed for constant exposure but did not achieve it |
|
| Chironomus sp. acute immobilisation test (OECD, 2011b) | In the control, including the solvent control if appropriate, not more than 15% of the larvae should show immobilisation or other signs of disease or other stress (e.g. abnormal appearance or unusual behaviour, such as trapping at the water surface) at the end of the test | This criterion is directly relevant as it demonstrates suitable conditions for the chironomids |
| The dissolved oxygen concentration at the end of the test should be ≥ 3 mg/L in control and test vessels | This criterion is directly relevant as it relates to ensuring suitable conditions | |
|
ALSO (not a validity criteria): The concentration of the test substance should be measured, as a minimum, in the control(s), the highest and lowest test concentration, but preferably in all treatments, at the beginning and end of the test. It is recommended that results be based on measured concentrations. However, if evidence is available to demonstrate that the concentration of the test substance has been satisfactorily maintained within ± 20% of the nominal or measured initial concentration throughout the test, then the results can be based on nominal or measured initial values. |
It is important that the test concentrations are measured, but the minimum requirements are likely to be greater for TKTD modelling as information is required on the whole exposure profile Maintaining constant exposure is not required to use the study for TKTD but the exposure profile should be described. This applies even if the study aimed for constant exposure but did not achieve it |
|
| Sediment‐water Chironomid toxicity test using spiked water (OECD, 2004b) | The emergence in the controls must be at least 70% at the end of the test | This criterion is directly relevant as it demonstrates suitable conditions for the chironomids |
| C. riparius and C. yoshimatsui emergence to adults from control vessels should occur between 12 and 23 days after their insertion into the vessels; for C. tentans, a period of 20–65 days is necessary | This criterion is directly relevant as it demonstrates suitable conditions for the chironomids | |
| At the end of the test, pH and the dissolved oxygen concentration should be measured in each vessel. The oxygen concentration should be at least 60% of the air saturation value (ASV) at the temperature used, and the pH of overlying water should be in the 6–9 range in all test vessels | This criterion is directly relevant as it relates to ensuring suitable conditions | |
| The water temperature should not differ by more than ±1.0°C. The water temperature could be controlled in an isothermal room and in that case the room temperature should be confirmed for appropriate time intervals |
This criterion is directly relevant as it relates to ensuring suitable conditions If a physiological model that includes variable temperature is used this could also be applied to the study |
|
|
ALSO (not a validity criteria): As a minimum, samples of the overlying water, the pore water and the sediment must be analysed at the start (preferably 1 h after application of test substance) and at the end of the test, at the highest concentration and a lower one |
It is important that the test concentrations are measured, but the minimum requirements in terms of frequency are likely to be greater for TKTD modelling as information is required on the whole exposure profile. Which compartments need to be measured will depend on the behaviour of the chemical and the model being used (i.e. if the model only uses water concentrations this should be the focus of the measurements | |
| Daphnia magna reproduction test (OECD, 2012) | The mortality of the parent animals (female Daphnia) does not exceed 20% at the end of the test. | This criterion is directly relevant as it demonstrates suitable conditions for the daphnids. |
| The mean number of living offspring produced per parent animal surviving at the end of the test is ≥ 60. | This criterion is directly relevant as it relates to ensuring suitable conditions. | |
|
ALSO (not a validity criteria): During the test, the concentrations of test substance are determined at regular intervals In semi‐static tests where the concentration of the test substance is expected to remain within ± 20% of the nominal, it is recommended that, as a minimum, the highest and lowest test concentrations be analysed when freshly prepared and at the time of renewal on one occasion during the first week of the test For tests where the concentration of the test substance is not expected to remain within ± 20% of the nominal, it is necessary to analyse all test concentrations, when freshly prepared and at renewal If a flow‐through test is used, a similar sampling regime to that described for semi‐static tests is appropriate (but measurement of ‘old’ solutions is not applicable in this case) |
It is important that the test concentrations are measured, but the requirements will be different for TKTD modelling as information is required on the whole exposure profile. Therefore the information in the guideline should be adapted to suit the experimental conditions and exposure profile being used Maintaining constant exposure is not required to use the study for TKTD but the exposure profile should be described. This applies even if the study aimed for constant exposure but did not achieve it |
|
| Freshwater alga and cyanobacteria growth inhibition test (OECD, 2011a) | The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72‐h test period. This corresponds to a specific growth rate of 0.92 day−1. For the most frequently used species the growth rate is usually substantially higher (see Annex 2 of the guideline). This criterion may not be met when species that grow slower than those listed in Annex 2 of the guideline are used. In this case, the test period should be extended to obtain at least a 16‐fold growth in control cultures, while the growth has to be exponential throughout the test period. The test period may be shortened to at least 48 hours to maintain unlimited, exponential growth during the test as long as the minimum multiplication factor of 16 is reached | This criterion is directly relevant as it demonstrates suitable conditions to allow sufficient growth are maintained. However, this criterion relates to the standard static study where low number of algae is inoculated allowing exponential growth. Since the study design will need to be different for time‐variable exposure studies, a more detailed consideration of how to determine suitable control performance will be needed for flow‐through systems |
| The mean coefficient of variation for section‐by‐section specific growth rates (days 0–1, 1–2 and 2–3, for 72‐hour tests) in the control cultures (See Annex 1 of the guideline under – coefficient of variation‖) must not exceed 35%. See paragraph 49 for the calculation of section‐by‐section specific growth rate. This criterion applies to the mean value of coefficients of variation calculated for replicate control cultures | This criterion helps to demonstrate that the controls do not experience increasing stress during the study, but that growth is stable and unlimited. A similar quality criterion should be set up for flow through systems, if they are to be used to test time‐variable exposures | |
| The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10% | This criterion is important because it helps to demonstrate that the controls are sufficiently similar to allow detection of differences in the treatment. A similar quality criterion could be applied for flow‐through systems monitoring control growth over time. The number of replicates and time‐points considered will, however, need reconsideration due to the different and more elaborate setup of flow‐through systems | |
|
ALSO (not a validity criteria): Provided an analytical procedure for determination of the test substance in the concentration range used is available, the test solutions should be analysed to verify the initial concentrations and maintenance of the exposure concentrations during the test Analysis of the concentration of the test substance at the start and end of the test of a low and high test concentration and a concentration around the expected EC50 may be sufficient where it is likely that exposure concentrations will vary less than 20% from nominal values during the test. Analysis of all test concentrations at the beginning and at the end of the test is recommended where concentrations are unlikely to remain within 80–120% of nominal. For volatile, unstable or strongly adsorbing test substances, additional samplings for analysis at 24 hour intervals during the exposure period are recommended in order to better define loss of the test substance |
It is important that the test concentrations are measured, but the requirements will be different for flow‐through systems as information is required on the whole exposure profile. Therefore, the information in the guideline should be adapted to suit the experimental conditions and exposure profile being used Maintaining constant exposure is not required to use the study for TKTD but the exposure profile should be described. This applies even if the study aimed for constant exposure but did not achieve it |
|
| Lemna sp. Growth inhibition test (OECD, 2006 ) | For the test to be valid, the doubling time of frond number in the control must be less than 2.5 days (60 h), corresponding to approximately a sevenfold increase in seven days and an average specific growth rate of 0.275 day−1. Using the media and test conditions described in this Guideline, this criterion can be attained using a static test regime. It is also anticipated that this criterion will be achievable under semi‐static and flow‐through test conditions. Calculation of the doubling time is shown in paragraph 49 | This criterion is directly relevant as it demonstrates suitable conditions to allow sufficient growth. Monitoring surface area/frond number on a daily bases allows checking if this criterion is met also during longer duration experiments where change of media and/or thinning is implemented |
|
ALSO (not a validity criteria): During the test, the concentrations of the test substance are determined at appropriate intervals. In static tests, the minimum requirement is to determine the concentrations at the beginning and at the end of the test In semi‐static tests where the concentration of the test substance is not expected to remain within ±20% of the nominal concentration, it is necessary to analyse all freshly prepared test solutions and the same solutions at each renewal If a flow‐through test is used, a similar sampling regime to that described for semi‐static tests, including analysis at the start, mid‐way through and at the end of the test, is appropriate, but measurement of ‘spent’ solutions is not appropriate in this case |
It is important that the test concentrations are measured, but the requirements will be different for TKTD modelling, as information is required on the whole exposure profile. Hence, for static systems more measuring times may have to be included to properly be able to model exposure profiles, and for semi‐static or pulse exposures this is even more important. Therefore the information in the guideline should be adapted to suit the experimental conditions and exposure profile being used Maintaining constant exposure is not required to use the study for TKTD but the exposure profile should be described. This applies even if the study aimed for constant exposure but did not achieve it |
|
| Sediment free Myriophyllum spicatum toxicity test (OECD, 2014a) | For the test to be valid, the doubling time of main shoot length in the control must be less than 14 days. Using the media and test conditions described in this Guideline, this criterion can be attained using a static or semi‐static test regime | This criterion is directly relevant as it demonstrates suitable conditions to allow sufficient growth are maintained |
| The mean coefficient of variation for yield based on measurements of shoot fresh weight (i.e. from test initiation to test termination) and the additional measurement variables (see paragraph 37 of this guideline) in the control cultures do not exceed 35% between replicates | This criterion is important because it helps to demonstrate that the controls are sufficiently similar to allow detection of differences in the treatment | |
| More than 50% of the replicates of the control group are kept sterile over the exposure period of 14 days, which means visibly free of colonisation by other organisms such as algae, fungi and bacteria (clear solution). Note: Guidance on how to assess sterility is provided in the ring‐test report referenced in the guideline | This criterion is important because colonisation by other organisms could affect the results, increasing variability between replicates and making it harder to detect effects | |
|
ALSO (not a validity criteria): During the test, the concentrations of the test substance(s) are determined at appropriate intervals. In static tests, the minimum requirement is to determine the concentrations at the beginning and at the end of the test In semi‐static tests where the concentrations of the test substance(s) are not expected to remain within ± 20% of the nominal concentration, it is necessary to analyse all freshly prepared test solutions and the same solutions at each renewal |
It is important that the test concentrations are measured, but the requirements will be different for TKTD modelling as information is required on the whole exposure profile. Therefore the information in the guideline should be adapted to suit the experimental conditions and exposure profile being used Maintaining constant exposure is not required to use the study for TKTD but the exposure profile should be described. This applies even if the study aimed for constant exposure but did not achieve it |
|
| Water sediment Myriophyllum spicatum toxicity test (OECD, 2014b) | For the test results to be valid, the mean total shoot length and mean total shoot fresh weight in control plants at least double during the exposure phase of the test. In addition, control plants must not show any visual symptoms of chlorosis and should be visibly free from contamination by other organisms such as algae and/or bacterial films on the plants, at the surface of the sediment and in the test medium | This criterion is directly relevant as it demonstrates suitable conditions to allow sufficient growth are maintained and because contamination could affect the results, increasing variability between replicates and making it harder to detect effects |
| The mean coefficient of variation for yield based on measurements of shoot fresh weight (i.e. from test initiation to test termination) in the control cultures does not exceed 35% between replicates | This criterion is important because it helps to demonstrate that the controls are sufficiently similar to allow detection of differences in the treatment | |
|
ALSO (not a validity criteria): The correct application of the test chemical should be supported by analytical measurements of test chemical concentrations Water samples should be collected for test chemical analysis shortly after test initiation (i.e. on the day of application for stable test chemicals or one hour after application for substances that are not stable) and at test termination for all test concentrations Concentrations in sediment and sediment pore‐water should be determined at test initiation and test termination, at least in the highest test concentration, unless the test substances are known to be stable in water (> 80% of nominal). Measurements in sediment and pore‐water might not be necessary if the partitioning of the test chemical between water and sediment has been clearly determined in a water/sediment study under comparable conditions (e.g. sediment to water ratio, application method, sediment type) See full information about analytical measurements in paragraphs 69–79 |
It is important that the test concentrations are measured, but the requirements will be different for TKTD modelling as information is required on the whole exposure profile. Therefore the information in the guideline should be adapted to suit the experimental conditions and exposure profile being used If a water sediment system is used this is likely to be extremely complicated for use in TKTD modelling because the pulses in the water and sediment will not coincide, so additional consideration of the route of exposure and internal transportation of chemicals between compartments is required Maintaining constant exposure is not required to use the study for TKTD but the exposure profile should be described. This applies even if the study aimed for constant exposure but did not achieve it |