There has been a shift in the procedure to perform the tier 1 aquatic risk assessment of pesticides for primary producers. In the past (i.e., when [4], was in force), the common practice was to select the lowest available endpoint among yield (EyC50), Area Under the Curve (AUC or “biomass”; EbC50) and growth rate (ErC50) endpoints; AUC and yield with EbC50 and EyC50 endpoints were in most cases the lowest. Nowadays - i.e., since the aquatic guidance document [6] was put in force- the endpoint recommended for tier 1 is the growth rate ErC50 endpoint. This change in the selection of the endpoint is justified since the growth rate ErC50 endpoint is a more robust estimate compared to AUC and yield (EbC50 or EyC50) endpoints. However, using this endpoint while keeping the same AF of 10 has significant implications on the protection level of primary producers. These aspects and implications are presented.
Change of endpoint selected and shift in conservatism of tier 1 assessment
To compare the protection levels set by the tier 1 risk assessment for primary producers, we used a large and representative EU regulatory dataset. We evaluated how the recommendation of using the growth rate ErC50 endpoint instead of the lowest endpoint (Area Under the Curve (AUC/ “biomass”) or yield, i.e. EbC50 or EyC50) has shifted the level of conservatism. To investigate this, we compared the ErC50 values with the Eb/yC50 values for algae and macrophytes.
Our results indicate a clear change since the tier 1 endpoint currently used (ErC50) is in average approximately 3.5 and 2.1 times higher than the endpoints previously used (EbC50 or EyC50) for algae and macrophytes, respectively.
For algae, the 90th percentiles of the ratios between the endpoint for growth rate and those for AUC and yield (i.e., ErC50/EbC50 and ErC50/EyC50 ratio) are 5.6 and 6.5, respectively. For macrophytes, these ratios are 3.2 and 4.0, respectively. The shift between the currently used growth rate endpoint and the previously used lowest endpoints is slightly larger when yield values are considered rather than AUC. The shift is also greater for algae compared to for macrophytes. Those shifts in ratios are consistent across different species tested within each group (i.e., algae and macrophytes).
These results indicate that the level of conservatism of the tier 1 risk assessment for aquatic primary producers has decreased since the growth rate inhibition endpoint (ErC50) is now being used instead of the lowest AUC (or ”biomass”) and yield endpoints (Eb/yC50). This confirms the results reported by Swarowsky et al. [18], who demonstrated that Eb/yC50 endpoints are consistently lower than the corresponding ErC50 endpoint (with the 90th percentiles of EyC50 values for algae and macrophytes being 7.8 and 3.5).
Our findings from the comparisons between growth rate inhibition and biomass-related endpoints based on a large dataset are supported by the derived mathematical relationships, which show that the inhibition observed in biomass surpasses that of the of growth rate and that these differences increases as growth rate and test duration increase. It is expected that the shifts between ErC50 and EyC50 are slightly higher compared to EbC50, while the substantial difference between algae and macrophytes can be attributed to significantly lower growth rates of macrophytes, having more influence than longer test durations. To derive the exact mathematical relationship between the growth rate inhibition endpoint (ErC50) and the lowest endpoint (Eb/yC50), additional test specific information regarding the dose-response slopes for biomass inhibition is required. Further insights into the mathematical relationship between biomass and growth-related endpoints can be found in [7]. The evident trend of systematically higher inhibitions in yield and AUC compared to the growth rate substantiates the disparity in the level of conservatism. Consequently, replacing the use of the lowest available endpoint (ExC50) with the growth rate inhibition endpoint (ErC50) while maintaining the same assessment factor (AF) of 10 when deriving the regulatory acceptable concentration (RACs) results in a strong shift to lower conservatism and a reduction in the level of protection set at tier 1. However, such reduction in protection level does not necessarily result in an unsuitably low protection level. In order to assess this aspect, we compared the protection levels achieved currently (based on the growth rate inhibition endpoint) and previously (based on the lowest endpoint) to a reference that is the tier 3 considered as surrogate reference tier, as discussed in the following.
Suitability of the tier 1 protection levels achieved when using different endpoints
In the comparison of protection levels achieved currently (i.e., with Tier-1 RAC based on the ErC50 growth rate inhibition endpoint) and previously (i.e., with Tier-1 RAC based on the lowest endpoint- Area Under the Curve (AUC/ “biomass”) or yield, i.e. Eb/yC50), we selected the data in a strict way to limit the uncertainties around the analysis. First, we performed our analysis on tier 1 data for active substance (a.s.) by using the current EFSA list of endpoints (LoEPs). By doing so, we ensure that the selected data are those used in EU-wide harmonized tier 1 risk assessment, and thus correspond to those setting the level of protection in the authorisation procedures of PPPs in the Member States. In a second step, we limited the dataset to those substances for which relevant tier 3 information is available (i.e., from model ecosystem experiments/ micro-mesocosm studies as published in the same EFSA LoEPs).
In the tiered approach of the aquatic risk assessment, assuming that tier 3 studies are an appropriate surrogate reference tier and the Tier-3 RAC values are appropriately set (i.e., not considerably under- or over-protective), it can then be assumed that Tier-1 RAC values lower than Tier-3 RAC values is indicative of a suitable minimum level of protection in tier 1 risk assessment. Inversely, Tier 1-RAC values higher than Tier-3 RAC values would indicate an under-protective tier 1 risk assessment. When comparing the achieved protection levels, we found that the current tier 1 RA based on ErC50 endpoints and an AF of 10 is more frequently insufficiently protective (i.e., in ca 45% of cases; Fig. 4A) than when based on Eb/yC50 endpoints with the AF of 10 used previously (i.e., in ca 10% of cases; Fig. 4B).
Hartmann et al. [19] conducted a similar evaluation, using a smaller dataset of 16 substances in total; among them, 14 substances were also included in our study. The authors concluded that the current and previous risk assessments provided "equal protectivity" in over 80% of cases. Our findings appear to contradict their outcomes. Our analysis indicates that 13 out of 20 substances remained in the same "category of protectiveness" (i.e., either above or below the 1:1 line as presented in Fig. 4) while this was shown for 14 out of 16 substances in Hartmann et al. [19]. Overall, our analysis indicates that only for 11 out of 20 substances in our dataset (55% of cases) a minimum protection level considered as suitable according to the SPGs is reached, compared to 11 out of 15 substances in [19] (74% of cases).
The minimum protection level considered as suitable is ensured when Tier-1 RACs are below Tier 3-RACs and this is currently achieved for a significantly lower number of cases than previously (11 versus 18 a.s. out of a total of 20 a.s.; i.e., 55% versus 90% of cases; Fig. 4A and B). Similar work by Duquesne et al. [14, 15] on slightly different datasets also showed that using the ErC50 instead of the Eb/yC50 resulted in a lower number of cases having a protective Tier 1 RA (i.e., around 40% vs. 75% of cases). However, in Hartmann et al. [19], the difference was not as large, with 74% of cases currently achieving such protection levels compared to 87% previously. Wjingaarden and Arts [13] also performed a similar evaluation and found that selecting ErC50 as the regulatory endpoint instead of the lowest endpoint, all with the standard AF of 10, generally maintained the protection level. They showed that using ErC50 endpoints resulted in protection levels considered as suitable for 80% of cases (i.e., for 8 out of 10 substances) compared to 75% of cases when using the lowest endpoints (i.e., for 3 out of 12 substances). However, there were differences in the data selection between their study and ours. They used a variety of sources, i.e., from draft assessment reports published by the EFSA and from the USEPA-ECOTOX database, whereas we restricted our data to the EFSA LoEPs, resulting in only 6 substances common to both datasets, which might explain the discrepancies between the conclusions
Our approach offers the advantage of defining the level of protection achieved specifically when applying the current regulation practice in Europe, leading to non-arbitrary decisions, especially when deriving higher Tier endpoints and the regulatory acceptable concentration RACs since these can be more open to interpretation than in the lower tier. The a.s. evaluation on EU-level involves various Member states and delivers expert agreed endpoints. Differences between our study and the studies of [19] and [13] include the fact that they exclusively selected substances with herbicidal mode of action, whereas we also considered fungicides to broaden the dataset; for some of these fungicides, primary producers are not the group most at risk but except for mancozeb, the higher tier data deliver regulatory acceptable concentrations also applicable for primary producers (i.e., for chlorothalonil and dodine). These authors also selected the Tier-3 RACs based only on the effect threshold option (ETO-RAC), whereas we selected the ERO-RAC when it delivered a more conservative value than the ETO-RAC (case of isoproturon, see Table S4) or when it was the only RAC available (i.e., frequent in older evaluations delivering only a NOAEC, cases of metamitron, metazachlor, acetochlor, iodosulfuron, lenacil and bifenox); the reason is that we consider that the lowest value sets the level of protection to be achieved in the field.
Bergtold and Dohmen [20] also performed a comparison of EbC50 and ErC50 endpoints for primary producers to assess their relevance for the aquatic RA of herbicides, referring to higher tier studies using the effect classes approach (e.g., [21, 66]). They concluded that using ErC50 values in combination with an AF of 10 is sufficient to exclude unacceptable risk to algae and aquatic plants in the environment. This finding diverges from our scientific evaluation.
Overall, different studies have been conducted to evaluate the protection level achieved for primary producers in the first tier risk assessment by comparing Tier-1 RACs and higher Tier RACs derived from microcosm and mesocosm studies. They indicate two different types of outcomes, i.e., a shift towards a lower protection level when using ErC50 (as observed in our study) versus a tendency for the protection level to be maintained (as observed in other studies). They also show that small changes in the data selection can lead to significant variations in outcomes, given the relatively limited datasets (with a maximum of 20 substances in our study). To address this issue and conduct a more robust analysis as well as to assess if the SPG “no population affected” is reached for each substance, we applied the concept of the cumulative distribution function. This analysis shows that the minimum protection level considered as suitable was insured in 87.7% of the cases previously versus 66.3% currently (Fig. 5A and B) which confirms the extent of the shift in the protection level achieved.
Should the protection level currently achieved be considered sufficient?
The EFSA Opinion on the development of specific protection goals for pesticide risk assessment [9] requires that new risk assessment methods should be calibrated to ensure that they meet the SPGs. While some member states have noted the aquatic guidance document [6], they have also called for calibration to be checked for primary producers following the change of the type of endpoint used in RA. Therefore, it is necessary to evaluate the consequences of the shift in the protection level achieved for primary producers resulting from the current use of ErC50 values with the default AF 10, in order to determine whether the SPGs are being met under the practice proposed in the aquatic guidance document [6], or if it should be adapted.
We question whether the current shift in the level of conservatism for the tier 1 risk assessment of primary producers is acceptable, since it is resulting in a protection level - considered as the minimum suitable - reached in 66.3% versus 87.7% of active substances when using Tier-1 RACs derived from the ErC50 instead of Eb/yC50 values. In other words, should such a protection level ensured only in ca 66% of the a.s. be considered sufficient, or should it be modified? To achieve the previous level reached for ca 88% of a.s., an AF of ca 24 instead of the default AF of 10 should be used for the current tier 1 RA. We are aware that these values may be approximative since our dataset is limited to a total of 20 substances; however, they are indicative of the correct trend. Our recommendation to increase the default AF to be applied to the growth rate endpoint ErC50 endpoint that was set by the Uniform principles [5] and to change the current EU risk assessment practice for primary producers differs from those of other authors who concluded that the current protection level remained sufficient. Another approach could be to consider using the growth rate endpoint equivalent to 10% inhibition (i.e., ErC10 instead of ErC50) and keep the default AF of 10. However, ExC10 endpoints are statistically less robust than ExC50 endpoints. Thus, this approach has important drawbacks and was not pursued.
Furthermore, it is important to question whether the previous minimum level of protection of the tier 1 risk assessment approach based on the lowest endpoint and an AF 10, which was reached for only ca 88% of a.s., was actually sufficient to protect populations and communities in the field. The approval of an a.s. for use in PPP after the EU evaluation implies that the SPGs are being met, and by consequence, that no unacceptable effects on populations will occur in the field for each group of organisms. Having a failing rate of approximately 12% of the a.s. is an issue to be aware of, especially when it is expected that the tier 1 risk assessment of each single a.s. is suitable to protect the primary producers. It seems essential to ensure a suitable level of protection of tier 1 RA, e.g., by increasing the AF values of ca 40 and 92 in the current situation based on the growth rate ErC50 endpoint (corresponding to ca 19 and 60 in the previous situation based on the lowest endpoint Eb/yC50) for achieving the aim of an adequate protection level reached in 95–99% of the active substances evaluated and PPPs authorised. Increasing the requirements to e.g. 95% of the cases is recommended. Indeed, it is crucial to maintain the function of primary producers since they are the lowest trophic level and have thus a fundamental role in the aquatic food web, and eventually to maintain and restore biodiversity of aquatic communities especially threatened in agricultural landscapes [22; 23].
In addition to this comparison and calibration between tiers related to the shift of endpoints selected in the tier 1 RA, it is also worth noting some general aspects and concerns of the aquatic risk assessment, also relevant in the context of the previous assessment based on the lowest endpoints. In the concept of the tiered approach, the lower tier risk assessment should be simpler and more conservative than the more complex and realistic higher tier risk assessment (e.g., [6]). The extent of this high conservatism to be ensured at tier 1 is intrinsically linked to the tier 3 RA. Indeed, there are uncertainties about the suitability of the protection level being actually achieved at tier 1 for borderline cases (i.e., close to 1:1 regression line between Tier-1 RACs and Tier-3 RACs) and this could be solved by clarifying the level of conservatism of the tier 3 risk assessment. This tier 3 aquatic RA is considered as a surrogate reference Tier for the field situation, which is the actual reference Tier. However, the final step of calibration, which involves the evaluation of the protectiveness of the assessment based on the surrogate reference tier compared to the reference tier, needs to be documented. This is a significant concern because the representativeness of the assemblages tested in micro-/mesocosm studies for real field conditions may be limited [24]. In addition, a large- scale monitoring study of surface waters in agricultural areas of Germany identified that the current authorisation of PPPs underestimates the actual ecological risk to invertebrates [25]. Therefore, it is crucial to collect further data from appropriate monitoring or field studies to verify that the prospective risk assessment is appropriately calibrated, and thus delivers suitable estimates that ensure sufficient protection for primary producers under field situation. As of now, there are uncertainties about the protection level achieved for algae and macrophytes when mesocosms are used as a surrogate for the field situation. It should be also noted that referring to the ErC50 is most suitable for species that have exponential growth such as algae and Lemna spp. as the calculation of inhibition of the average specific growth rate is based on logarithmic transformation. For some other species of macrophytes that do not present an exponential growth, the growth rate endpoint ErC50 is less suitable and may lead to an underestimation of the risk due to PPP exposure although it is however more robust than the biomass- related endpoints (Eb/yC50) when considering varying test conditions.
These aspects indicate a lower degree of conservatism and various types of uncertainties when assessing the minimum level of protection to be actually achieved in the field. Hence, it is important to acknowledge them while conducting risk assessments; this is even emphasized under the current practice of using the ErC50 and thus less conservative endpoints in the ERA.