EFPIA Comments to the Draft WFD Dossier for Ibuprofen
Upon review of the draft WFD dossier prepared for ibuprofen, the European Federation of Pharmaceutical Industries and Associations (EFPIA) offer the following comments and analyses.
Below we present analyses of the measured concentrations data (Dossier Section 6.2) and the aquatic ecotoxicitydata (Dossier Section 7.1). In addition, we propose an alternative AA-QS. Given the abundant data set available for ibuprofen we assert that the data be considered through probabilisticas opposed to deterministic methods.
With regard to environmental concentrations, we present the work of two authors who have used statistical analyses of multiple measurements to propose what can be expected to be an ‘overall’ value which we believe to be a more pragmatic ‘Continental’ estimate for use in risk assessment.
Similarly, we have combined the wealth of toxicity data in a Species Sensitivity Distribution and are confident that the resulting HC5 (95%protective concentration) of 17.6 mcg/L and LCL (Lower Confidence Limit) of 1.1 mcg/L support the contention that an AA-QS of 1 mcg/L is robust and protective.
Below are details of both analyses along with citations. We commend the Commission and Rapporteur for the transparency with which this exercise has been managed and look forward to further dialog.
Comment to Section 6.2 - Measured concentrations
Measured Environmental Concentrations (MECs) of ibuprofen have been reported for various European water bodies. Comprehensive reviews of these data have been conducted by Straub (2006)[1] and Fent et al. (2006)[2]. Straub (2006) collected 311 individual European MECs reported in the literature from 1996 to 2006. MECs ranged from 0.001 to 5 µg l-1, although the vast majority of the data were below 1 µg l-1. Using a combined regression based on all available data (including the Weigel et al datum identified in the current Dossier Section 6.2 for the freshwater MEC) the author calculated an overall median surface water MEC for ibuprofen of just below 0.01 µg l-1 and a 90th percentile (MEC90) of approximately 0.185 µg l-1.
Fent et al. (2006) conducted a similar analysis with ibuprofen MEC data from Europe and the USA published between 1998 and 2005. However, rather than using individual values, the median, mean and maximum MECs from each of the reviewed studies were plotted. The data ranged from approximately 0.01 to 8 µg l-1, but again the vast majority of the data were below 1 µg l-1. The bulk of the median and mean values fell between 0.01 and 0.1 µg l-1. MECs above 0.1 µg l-1 tended to be maximum reported concentrations. These reviews show that in European surface waters the mean and median MECs for ibuprofen tend to fall between 0.01 and 0.1 µg l-1 and that an overall European median surface water MEC for ibuprofen is close to 0.01 µg l-1. MECs of 1 µg l-1 or more are likely to occur only in a very small fraction of heavily polluted sites such as stormwater canals
References
Fent K. Weston A., Caminada D. (2006) Ecotoxicology of human pharmaceuticals Aquatic Toxicology 76 (2006) 122–159
Straub J. (2006) Matters of fact and of fiction: European PECs and MECs of Ibuprofen. Poster, SETAC Europe 16th Annual Meeting, The Hague.
Comment to Section 7. - Effects and Quality Standards
Acute and chronic data are available for ibuprofen, including many micro-organism minimal inhibitory concentrations. [IUCLID, Chowdhury et al. 1996, Sanyal et al. 1993]. There is no explicit information on ibuprofen being inhibitory or not to sewage sludge micro-organisms in the biodegradation tests. However, as significant degradation was reached in both ready tests [both cited in IUCLID], it is assumed that the applied concentration of 20 mg/L was not inhibitory. In view of the comparatively high concentrations in all tests, no disruption of the biological step in sewage works is expected, nor is there an indication for toxicity towards micro-organisms in the environment. In freshwater cyanobacteria (Synechocystis species), ibuprofen at 1 µg/l up to 1 mg/l (the highest tested concentration) stimulated growth as compared to controls and therefore was certainly not inhibitory [Pomati et al. 2004].
Acute Toxicity Data
For the marine diatom, Skeletonema costatum, an EC50 of 7.1 mg/L was reported in a 96-hr study, and an EC50 of 39.9 mg/L with a NOEC of 20.5 mg/L was reported for a 5 day exposure with a 9 day recovery period [cited in IUCLID].
Ibuprofen showed moderate to low toxicity against freshwater green algae and no obvious difference between the sodium salt and the acid. Two studies reported 72-hr EC50 values of 315 and 342 mg/L for Desmodesmus subspicatus [Cleuvers, 2003, Cleuvers, 2004]. There is also a 72-hr EC05 of 72.9 mg/L based on growth rate, which is analogous to a NOEC [Cleuvers 2004] and a 72-hr IC25 of >32 ug/L with a 10 ug/L NOEC in Selenastrum capricornutum based on a decrease in biomass [Brun et al. 2006]. When this latter value is compared to the 96-hour IC50 value of >30 mg/L for Selenastrum capricornutum [cited in IUCLID], the difference in effect cannot be explained.
In several acute ecotoxicity tests with invertebrates, ibuprofen had 48-hour EC50 values of 9.06–132.6 mg/L and NOECs of 3–66 mg/L in Daphnia magna [IUCLID, Cleuvers 2004, Han et al. 2006, Cleuvers 2003]. Two EC50 values of approximately 10 mg/L are cited in IUCLID but the original data cannot be assessed. The other four studies [Heckmann et al. 2005, Han et al. 2006, Cleuvers 2003 and 2004] are more recent, follow international protocols and have EC50 values consistently >100 mg/L. An acute test with the freshwater snail, Planorbis carinatus, resulted in a 96-hour LC50 of 17.1 mg/L [Pounds et al. 2004]. There is one marine invertebrate test, with a 96-hour EC50 >100 mg/L and a NOEC of 30 mg/L in Mysidopsis (Americamysis) bahia [cited in IUCLID]. Based on these data, ibuprofen is of low toxicity to invertebrates.
Acute, 96-hour toxicity of ibuprofen to fish is given by two citations in IUCLID. A LC50 of 173 mg/L and a NOEC of 10 mg/L was reported for the freshwater sunfish, Lepomis macrochirus, and a study with the estuarine species, Cyprinodon variegates, resulted in an LC50 >300 mg/L and a NOEC of 300 mg/L. A 48-hr LC50 of 23.9 mg/L was recently reported for Danio rerio embryos (Kehrer and Nagel, 2006). Based on these data, ibuprofen is of low acute toxicity to fish.
Chronic Toxicity Data
Three publications on toxicity to macrophytes give 7-day EC50 values to Lemna gibba of >1 mg/L [Brain et al., 2004], and an extrapolated 4 mg/L[Pomati et al. 2004] and a measured 22 mg/L in Lemna minor [Cleuvers, 2003]. These values are in reasonable agreement, but Brain and colleagues noted an EC10 >1 mg/l (and a NOEC of 1 mg/L)[Brain et al. 2004], while Pomati and co-workers set their NOEC or LOEC at a very low 1 µg/L based on an observation that growth was 14% lower than control on day 7 at 1 ug/L [Pomati et al. 2004]. Pomati has a very flat dose-response, and their no-effect level was not a statistical no-effect level, since their statistical section just describes probit analysis. The coefficient of variation was not presented so it is impossible to determine if that is a real NOEC. Cleuvers did not indicate a NOEC [Cleuvers, 2003]. Brain et al. changed the media daily, used five different endpoints (wet weight, frond number, chlorophyll a, chlorophyll b and carotenoids) and consistently found no significant effect of ibuprofen at 1 mg/L, the highest treatment level. Pomati and colleagues replaced the test solutions just once on day 5, used only frond number as the endpoint and found a decrease of 25% compared to controls at 1 mg/l, the highest treatment level. Cleuvers used five concentrations, 1, 3.2, 10, 32 and 100 mg/L, did not change the media, used number and area of fronds as the endpoints and did not report any untoward effects at lower concentrations. In view of daily media exchanges and more endpoints studied by Brain and co-workers, making for the most consistent exposure and for the most sensitive effects evaluation, and in view of no significant adverse effects seen by Cleuvers at 1 mg/L, the NOEC of 1 mg/L in the study by Brain et al. is the most robust for Lemna.
In a recent battery of five basic mode-of-action test systems with Ibuprofen using photosynthetic bacteria, green algae and genetically modified yeasts and Escherichia coli bacteria, there was no indication or evidence for any general, specific, unspecific or reactive toxicity other than minimal (narcotic or baseline) toxicity [Escher et al. 2005]. The strongest effect found was an inhibition of photosystem II quantum yield in algae at an EC50 of 92.1 mg/L, which falls in the reported range of algal effects data. Overall, this result supports a relatively low level of toxicity of ibuprofen, which in turn supports choosing the higher chronic NOEC for Lemna.
Chronic survival NOECs of 20 -33 mg/L (21-d) [Han 2006; Han 2010] and 40 mg/L (12-d) [Heckmann 2005] were reported for Daphnia magna. A boundary estimate of >32 ug/L (the highest concentration tested) was observed in a 7-d Ceriodaphnia study (Brun et al. 2006). The NOEC based on survival of M. macrocopa was >50.0 mg/L (Han et al. 2010).
After 21 d exposure with D. magna, the NOEC based on survival of the initial neonate was 33.3 mg/L, while the LOEC based on reproduction was 1.23 mg/L which was the lowest concentration tested. The PGR decreased but the magnitude of the effect was not expected to
result in negative population growth [Han 2010]. However, a previous study by Han [2006] reported a reproduction NOEC of 20 mg/L. Further, a reproduction NOEC of 10 mg/L has been reported [Heckmann 2005]. The NOEC for reproduction of M. macrocopa was 25 mg/L [Han 2010].
A chronic test with the freshwater snail, Planorbis carinatus, resulted in a 21-day NOEC of 1.02 mg/L with a LOEC of 2.43 mg/L [Pounds et al. 2004].
Algal NOECs of 72.9 mg/L (Cleuvers, 2004), >30 mg/L (IUCLID) and 10 ug/L (Brun et al. 2006) have been reported. A Skeletonema NOEC of 20.5 mg/L was reported [IUCLID].
Two chronic fish NOECs were reported for medaka (Flilppin 2007; Han 2010) and are discussed below.
Flippin et al (2007) evaluated effects of ibuprofen on Oryzias latipes (Japanese medaka) exposed for 6 weeks via water to three concentrations of ibuprofen (1–100 ug/L nominal concentrations). Reproductive parameters, including frequency of spawning, fecundity, egg size, and rate of fertilization, were measured for each pair of adult medaka following 6 weeks of exposure. Increasing exposure to ibuprofen significantly increased the number of eggs per reproductive event, but decreased the number of spawning events per week. The total number of eggs produced by pairs over the week of assessment did not differ with exposure (Kruskal–Wallis test, H= 1.792, p = 0.62). As ibuprofen treatment increased, the frequency of egg production (eggs/day) decreased at 100 ug/L. The number of days with eggs decreased at 10 ug/L. On days when they did reproduce, pairs exposed to 100 ug/L of ibuprofen produced nearly twice as many eggs as those in the control group. In all cases, the rate of fertilization was generally greater than 90% for all treatment groups; no difference was observed in the rate of fertilization between treatment groups (Kruskal–Wallis test, H= 2.278, p = 0.52). No pathological damage was evident the in the gills, livers and head kidneys of animals from the highest exposure group. A NOEC of 10 to 100 ug/L can be derived from this study.
Han et al (2010) evaluated effects of ibuprofen on O. latipes at concentration up to 1000 ug/L for 144 days. The authors report a NOEC for survival at 0.1 ug/L. Examination of the data indicates no consistent dose response for a number of parameters and a NOEC is more appropriately in the range of 1 to 10 ug/L based on survival, number of eggs per brood, and time to hatch of fertilized eggs. This study raises an important question: Since no lethality observed in fry or juveniles, why do adults have a 1000-fold lower NOEC than the juveniles? This result is in stark contrast to what was observed by Flippin, and suggests the study be replicated before it is used as a basis for setting a PNEC.
There are no explicit data available on the acute or chronic ecotoxicity of the main metabolites of ibuprofen, Ibu-OH and Ibu-CX.
In conclusion, acute toxicity values of ibuprofen range from 7 to over 300 mg/L while chronic algal, Lemna, daphnid, snail, fish, and Hydra NOECs have a much wider range between a possible, very low 1 µg/L and over 100 mg/L. The chronic NOECs for Lemna, snails, and Hydra are consistent at 1 mg/L. There is no evidence in a battery of 5 basic mode-of-action test systems for any toxicity other than baseline toxicity/narcosis; no evidence for specific or unspecific toxicity in a photosystem II inhibition test; no evidence for estrogen receptor-mediated toxicity; and no evidence for reactive toxicity in comparison of glutathione present/absent and DNA repair system present/absent micro-organisms [Escher et al., 2005]. The overall picture that emerges from these studies is the ecotoxicity potential of ibuprofen is a minor concern.
Proposed Ibuprofen AA-QS Derivation
In light of the robust chronic dataset available for Ibuprofen, a Species Sensitivity Distribution (SSD) can be constructed as outlined in the Technical Guidance Document to statistically derive a PNEC using all of the data that exist.
Given there are chronic studies in 11 species spread over multiple taxa, a SSD was constructed to derive the HC5 of 17.6 ug/L. The chronic data SSD id presented in the figure. The 95% Lower Confidence Limit of the HC5 was calculated according to Aldenberg and Slob (1993), where: log HC05 = mean - k x SD where k is based on sample size; mean and SD are from natural log of (GM NOECs). The 95% LCL of 1.1 ug/L is rounded to 1 ug/L and proposed as the PNEC.
The PNEC of 1 ug/L is predicted to be sufficiently protective of aquatic life. The use of the LCL rather than the HC5 adds conservatism to address questions of the completeness of the dataset. Further, due to the cyclical nature of exposure and measured environmental concentrations orders of magnitude lower than this value, additional levels of safety are inherent in this value. Therefore, this SSD-generated PNEC can be adopted as a protective AA-QS without application of an additional Assessment Factor.
PNECwater, chronic = 1 ug/L = AA-QS
Species Sensitivity Distribution for Ibuprofen
Percentiles, ug/L
HC05, 0.05 / HC05,95% LCL / 0.10 / 0.25 / 0.5017.6 / 1.1 / 50.6 / 294 / 2,080
Ibuprofen, CAS No. 15687-27-1
Acute Studies
Organism / Benchmarkug/L / Duration of Study / Reference / Comments
algae
S. capricornutum / >32 / 72-hr / Brun / EC25 Highest conc. tested
S. capricornutum / >30,000 / 96-hr / H-S; IUCLID / NEL
D. subspicatus / 315,000 / 72-hr / Cleuvers 2003 / EC50
D. subspicatus / 342,200 / 72-hr / Cleuvers 2004 / EC50
S. costatum / 7,100 / 96-hr / H-S; IUCLID / EC50 (saltwater diatom)
S. costatum / 39,900 / 5-d / H-S; IUCLID / EC50 (NOEC 20,500 ug/L)
H. attenuata / 25,000 / 96-hr / Quinn 2008a / LC50 Adult survival
H. attenuata / 1,000 / 96-hr / Quinn 2008b / NOEC fetal development
crustacean
D. magna / 9,060 / 48-hr / H-S; IUCLID / EC50
D. magna / 11,500 / 48-hr / IUCLID / EC50
D. magna / >32 / 48-hr / Brun / LC50
D. magna / 107,700 / 48-hr / Heckmann / LC50
D. magna / 132,600 / 48-hr / Han / LC50
D. magna / 108,000 / 48-hr / Cleuvers 2003 / EC50
D. magna / 101,200 / 48-hr / Cleuvers 2004 / EC50
M. bahia / >100,000 / 96-hr / H-S; IUCLID / EC50 (NOEC of 30,000 ug/L)
P. carinatus / 17,100 / 96-hr / Pounds / EC50
fish
D. rerio / 23,900 / 48-hr / Kehrer / LC50
L. machrochirus / 173,000 / 96-hr / H-S; IUCLID / LC50 (NOEC 10,000 ug/L)
C. variegatus / >300,000 / 96-hr / H-S; IUCLID / NEL
Chronic studies
Note: * indicates value not used in SSD; reason in comments
Organism / Benchmarkug/L / Duration of Study / Reference / Comments
S. capricornutum / 10 / 72-hr / Brun 2006 / NOEC (32 ug/L =LOEC)
S. capricornutum / >30,000 / 96-hr / IUCLID / No Effect Level
D. subspicatus / 72,900 / 72-hr / Cleuvers 2004 / EC05 growth
EC50 = 342,000
Skeletonema costatum / 20,500 / 5-d / IUCLID / EC50 = 39,900 ug/L
L. gibba / 1,000 / 7-d / Brain 2004 / NOEC
L. minor / 1,000 / 7-d / Pomati 2004 / NOEC
EC50 = 4,000 ug/L?
L. minor / 22,000* / 7-d / Cleuvers 2003 / EC50 (NOEC not indicated)
H. vulgaris / 1,000 / 7-d / Pascoe / NOEC - survival
H. vulgaris / 1,000 / 17-d / Pascoe / NOEC – feeding & budding
10 ug/L* NOEC for polyp regeneration (only conc.)
D. magna / 108,000* / 7-d / Cleuvers 2003 / EC50 (NOEC not indicated)
D. magna / 40,000 / 12-d / Heckmann / NOEC - survival
D. magna / 10,000 / 12-d / Heckmann / NOEC - growth
D. magna / 10,000 / 12-d / Heckmann / NOEC - reproduction
D. magna / 20,000 / 21-d / Han 2006 / NOEC - survival
D. magna / 20,000 / 21-d / Han 2006 / NOEC - reproduction
D. magna / 33,300 / 21-d / Han 2010 / NOEC – survival
D. magna / 1,230 / 21-d / Han 2010 / NOEC <1,230 ug/L for reproduction
C. dubia / >32* / 7-d / Brun 2006 / NOEC (EC25) highest conc.
M. macrocopa / 50,000 / 7-d / Han 2010 / NOEC – survival
M. macrocopa / 25,000 / 7-d / Han 2010 / NOEC - reproduction
P. carinatus / 5,360 / 21-d / Pounds 2008 / NOEC - survival
P. carinatus / 2,430 / 21-d / Pounds 2008 / NOEC - hatch
P. carinatus / 1,020 / 21-d / Pounds 2008 / NOEC - growth
O. latiptes / 100 / 6-wk / Flippin 2007 / NOEC- survival
O. latiptes / 10 / Flippin 2007 / NOEC - reproduction
O. latipes / 0.1 / 120-d / Han 2010 / NOEC - survival
O. latipes / 1 / Han 2010 / NOEC - reproduction
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[1]Straub JO. 2006. Matters of fact and of fiction: European PECs and MECs of ibuprofen. Poster presentation at SETAC Europe 16th Annual Meeting, The Hague, Netherlands, May 7-11, 2006.
[2] Fent K., Weston A.A. and Caminada D. (2006). Ecotoxicology of human pharmaceuticals. Aquatic Toxicology 76: 122-159.