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Long-term toxicity to fish

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Description of key information

The 21-d NOEC is taken as 62 mg/l in the freshwater fish Pimephales promelas.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
62 mg/L

Additional information

One chronic guideline test is available, an early life stage test (ELS) with eggs and larvae/fry of fathead minnow (Pimephales promelas) (ENSR, 1999b). The 31-d NOEC is 299 mg/l. . A non-guideline early life stage study by Moreels et al. (2000a) with eggs from the African catfish (Clarias gariepinus) is not deemed reliable or relevant for hazard or risk assessment purposes due to some major deficiencies in the study. In particular, average exposure concentrations have been incorrectly estimated.

A fish sexual development toxicity assay in accordance with the OECD 234 guideline is also underway the full results of which will be included in the dossier when available.

There are several studies examining potential effects of MTBE on reproduction of fish. A non-guideline study by Moreels et al. (2006b) on zebrafish (Danio rerio) is not deemed to be reliable due to a large number of limitations of the study, as described in the IUCLID study endpoint record. A guideline GLP study has been unable to reproduce the observed changes in male VTG level in zebrafish reported by Moreels et al. (2006b) (discussed below). A separate guideline GLP study with fathead minnow (Pimephales promelas) also did not yield any similiar findings on VTG (also discussed below). Other than the reported effect on VTG, Moreels et al. (2006b) also reported changes in sperm motility. However, there are were no effects on fertilization success in the study or in other fish reproduction studies. Furthermore, there are no concentration-response relationships evident in the study by Moreels et al (2006b), no historical database comparison to give further information on normal biological ranges and limited information on the procedure elements of the measurements. As sperm velocity measures are highly susceptible to timing with significant losses in velocity occurring within seconds after activation of the sperm (Kime et al. 1996), randomization of samples for measurement can be important.

Two short term reproduction assays are available on the toxicity of the test substance in zebrafish and the fathead minnow. Both studies were performed under GLP conditions and in accordance with test guidelines OECD 229 and U.S. EPA OPPTS 890.1350.

In the first study (Wildlife International Ltd, 2012) an exceptionally high exposure concentration of 147 mg/L was included in the study so that a comparison could be made with a study reported in the literature by Moreels et al. (2006b). Under usual circumstances, testing according to OECD 229 and U.S. EPA OPPTS 890.1350 test guidelines would not include such high exposure concentrations.

In the first study (Wildlife International Ltd, 2012), breeding groups of zebrafish were exposed to test substance at mean measured concentrations of 0.122, 3.04 and 147 mg/L for 21 days. The endpoints evaluated, to determine if the test substance might interact with the estrogenic or androgenic hormones axes of fish, were fecundity, fertility, plasma VTG levels, and gonad histopathology. In addition, survival, body length, and wet weight were measured as general indicators of toxicity. Fish exposed to treatment levels of 0.122 and 3.04 mg/L showed no effect on any of the endpoints measured except for a significant elevation in plasma VTG levels in male fish exposed to 3.04 mg/L. This elevation was considered to be marginal (3.4-fold) with individual values comparable to those normally observed in adult male zebrafish. Exposure of fish to test substance at 147 mg/L significantly reduced the total number of eggs produced and the number of eggs produced per female per reproductive day. This reduction in fecundity was accompanied by a significant increase in the incidence of oocyte atresia along with a significant increase in the accumulation of oocyte debris in the oviduct. Under the conditions of the test, results indicate that exposure to the test substance at 147 mg/L impairs reproductive output of females; consequently the NOEC for reproduction was determined to be 3.04 mg/L, the next exposure concentration tested.

No supporting evidence was found to indicate that theis effect on fecundity seen in this guideline study with zebrafish is due to disruption of endocrine regulatory processes. As noted by experts on the short term fish reproduction assay, changes in histopathology and fecundity without changes in more diagnostic endpoints are likely due more to general toxicity rather than an impact on endocrine modulation (Ankley and Jensen, 2014). Furthermore, the OECD Guidance Document 150 (OECD, 2012) on evaluating chemicals for their potential for endocrine disruption considers effects solely on fecundity as more indicative of systemic toxicity, especially at higher test concentrations. Similarly, oocyte .atresia, which can be a natural degenerative process (Guraya, 1993) that occurs spontaneously in zebrafish (Rossteuscher et al., 2008; Owens, 2007; OECD, 2006), can be indicative of having reached a systemic level of toxicity (McCormick et al., 1989).

Based on the endpoints evaluated, it can be concluded that MTBE does not appear to interact with the estrogenic orandrogenic hormone axes of zebrafish, as discussed in a recent publication by Mihaich et al. (2015). The results from Moreels et al. (2006b) on VTG, which had reported an approx. 26 fold increase in male zebrafish VTG after a 3-week exposure to MTBE at 0.11 and 37 mg/L,were clearly not reproducible in the guideline study with zebrafish (Wildlife International Ltd., 2012). A detailed comparison of VTG results from these two zebrafish studies has been discussed by Mihaich et al. (2015), which highlights several uncertainties and limitations in the VTG levels reported by Moreels et al. (2006b), as well as the lack of reporting on whether the mid-exposure group of 2.7 mg/L had any difference to controls.

Due to the low NOEC identified in the guideline zebrafish study based on the apical endpoint of fertility, as well as and the slight variation in VTG levels in males, a second guideline study was planned in zebrafish using more test concentrations. However, an infection of nematodes was evident in the fish, potentially including zebrafish stocks available to the lab. As the fathead minnows (Pimephales promelas) are the more common test organism for the endocrine screening assay, and fathead minnows can also display secondary sex characteristics whereas the zebrafish does not possess quantifiable secondary sex characteristics, a follow-up study was undertaken with fathead minnows.

In this second guideline short-term reproduction study (Wildlife International Ltd, 2013) breeding groups of fathead minnows were exposed to test substance at mean measured concentrations of 0.0, 1.8, 6.2, 20 and 62 mg/L for 21 days. Under the conditions of the test, there were no apparent effects on survival, growth, reproduction, secondary sex characteristics, GSI, VTG or gonad histopathology in male or female fish exposed to the test substance for 21 days. A NOEC for reproduction was determined to be 62 mg/L, the highest exposure concentration tested. Based on the endpoints evaluated, MTBE does not appear to interact with the estrogenic or androgenic hormone axes of fathead minnows.

Based on research by Bonventre et al. (2011, 2012), MTBEmay target developing bIood vessels. In zebrafish, dose dependent increases of pooled blood in the common cardinal vein, cranial hemorrhages and abnormal intersegmental vessels were observed. The study has several limitations, such as no information on oxygen levels in the test system, even though some reported effects could be caused by oxygen depletion. 

 

If MTBE acts on the developing vasculature, then effects would be evident in the fish early life stage study. It is possible that anti-angiogenesis is the mode of action for effects on growth rate. The nominal NOEC of 255 mg/L (2.5 mM) for anti-angiogenesis in zebrafish is around the NOEC of 299 mg/L for fathead minnows. The relevance of the observed effects at 440 mg/L by Bonventre et al. (2011, 2012) is however limited, as such high concentrations greatly exceed OECD test guideline recommended maximal exposures for short-term toxicity testing on embryo and sac-fry stages. Such concentrations have a very potential limited relevance to MTBE in the aquatic environment for long-term toxicity and PNEC derivation.

 

With the existing database, a NOEC of 3.04 does not adequately characterize or quantify the potential toxicity of MTBE to fish due to the spacing of the exposure concentrations used in the study. The NOEC of 62mg/L is a more relevant point of departure, even though that test concentration is still above the maximum recommended limit concentration of 10 mg/L in the OECD 229 test guideline.

The fish endocrine screening studies are intended to be used for qualitative risk assessment purposes, such as to determine potential for endocrine interation and further testing. As the OECD Fish Toxicity Framework (OECD, 2012) considers that dose spacing greater than a factor of around 3 should be considered for qualitative rather than quantitative risk assessment purposes, the NOEC for fish is considered as 62 mg/L rather than 3.04 mg/L. Considering the NOEC of 3.04 as not sufficiently precise an indication of toxicity to fish be relevant for quantitative risk assessment purposes is further supported by the next exposure concentration of 147 mg/L greatly exceeding the max. concentration for testing under the OECD 229 guideline of 10 mg/L. The U.S. EPA considers its corresponding OPPTS 890.1350 guideline with a max. concentration for testing of 100 mg/L as not intended to provide quantitative risk assessment data (U.S. EPA, 2008).

References: 

Ankley, GT and Jensen, KM (2014). A novel framework for interpretation of data from the fish short-term reproduction assay (FSTRA) for the detection of endocrine-disrupting chemicals. Environ Toxicol Chem 33(11), 2529-2540.

 

Guraya, SS (1993). Follicular Atresia. Proc Indian Nat Sci Acad 39B(3), 311-332.

 

McCormick, JH, Stokes, GN and Hermanutz, RO (1989). Oocyte atresia and reproductive success in fathead minnows (Pimephales promelas) exposed to acidified hardwater environments. Arch Environ Contam Toxicol 18, 207-214.

 

Mihaich, EM, Erler, S, LeBlanc, G and Gallagher, S (2015). Short term fish reproduction assays with methyl tertiary butyl ether (MTBE) with zebrafish and fathead minnow: Implications for evaluation of potential for endocrine activity. Environ Toxicol Chem. Epub ahead of print available at DOI 10.1002/etc.3017

 

OECD (2006). Report of the initial work towards the validation of the 21-day fish screening assay for the detection of endocrine active substances (Phase 1A). Series on Testing an Assessment Number 60. NV/JM/MONO(2006)27. Paris, France.

 

Owens, JW (2007). Phase-2 OECD 21-day Fish Screening Assay Validation Report. CEFIC Negative Test Substance Studies. Report of Three 21-day Fish Endocrine Screening Assays to Complete CEFIC’s Contribution to Phase-2 of the OECD Validation Program: Studies with Potassium Permanganate, n-Octanol, and 2-Methoxyethanol in the Fathead Minnow (Pimephales promelas). OECD, Paris, France.

 

Rossteuscher, S, Schmidt-Posthaus, H, Schaefers, C, Teigeler, M and Segner, H (2008). Background pathology of the ovary in a laboratory population of zebrafish Danio rerio. Dis Aquat Org 79, 169–172.