Registration Dossier

Ecotoxicological information

Endpoint summary

Administrative data

Description of key information

Due to the low water solubility the studies (OECD 201, OECD 202, OECD 203) were carried out with Water Accommodated Fractions (WAF). Where test concentrations of the test item were measured they were below 1 mg/L. Results were expressed based on the loading rates.

Guideline

Test species

Endpoint

Result, mg/L loading
rate (Measured)

Remarks

OECD 203

Cyprinus carpio

96h-LC50

> 100 (Meas: 0.7- 0.075)

Key study
GLP, rel.1

OECD 203

Danio rerio

96h-LC50

(Meas.) 1216.1 mg/L
**

Supporting study
GLP, rel. 1

OECD 234

Danio rerio

63d-NOEC

63d-LOEC

<46.9 µg/L (Meas.)

46.9 µg/L (Meas.)

Key study

GLP, rel. 1

OECD 210 (modified)

Danio rerio

21d-NOEC

≥30 µg/L (Meas.)

Supporting study

non-GLP, rel.1

OECD 202

Daphnia magna

48h-EC 50

> 100 mg/L
(Meas: >0.0271 mg/L)

Key study

GLP, rel. 1

OECD 201

Pseudokirchneriella
subcapitata

*72h- NOEC growth
*72h-EC50 growth

> 32 mg/L

> 100 mg/L
(Meas. < 0.001)

Key study
GLP, rel.1

OECD 201

Scenedesmus subspicatus
= Desmodesmus
subspicatus

72h-EC10
72h-EC50

> 100 mg/L

> 100 mg/L
Not measured

Supporting study
GLP, rel. 1

OECD 209

Activated sludge (domestic)

30 min-NOEC

> 1000 mg/L
Not measured

Key study

no GLP, rel. 1

OECD 231

Xenopus laevis

21d-NOEC

≥44.2 µg/L (Meas.)

Key study

GLP, rel. 1

* 24-h Pre-exposure period in the dark in view of photodegradability of the test substance. Test conc. after 96 h (is after 72 h of light) < 0.001 mg/L.
** This measured concentration is far above the water solubility limit.

Additional information

Endocrine disrupter (ED) assessment

Two studies were conducted in order to assess the endocrine disruptive properties of the test item - OECD 231 The Amphibian Metamorphosis Assay and OECD 234 Fish Sexual Development Test (see IUCLID Section 6.1.8). Both studies were GLP-conform and all acceptance criteria were fulfilled, therefore, both studies are considered to be valid.

The OECD 231 study was performed with the test substance in which Nieuwkoop and Faber (NF) stage 51 Xenopus laevis larvae were exposed to three (3) different concentrations of the test chemical (n = 4 replicates per concentration) and dilution water control (n = 4 replicates) for 21-days. The Amphibian Metamorphosis Assay (AMA) is a screening assay intended to empirically identify substances which may interfere with the normal function of the hypothalamic-pituitary-thyroid (HPT) axis.  Prior to in-life testing, the limit aqueous solubility was determined at test conditions based on general guidance provided by OECD No. 105. Results of practical water solubility analyses of centrifuged samples from the recirculating saturator columns indicated that the mean concentration based on the three consecutive centrifuged samples was 48.1 µg/L OMC. The difference between each of the three consecutive samples relative to the mean ranged from 2.1-7.1%. The difference between centrifuged and uncentrifuged samples for the three consecutive samples ranged from 2.8-6.3%. 

Based on the determined limit aqueous solubility under test conditions, the target test item concentrations selected for the Amphibian Metamorphosis Assay study were 0.0 (control), 5.50, 16.5, and 50 µg/L. Larval density at test initiation was 20 tadpoles per test tank (replicate). A flow-through diluter system (Benoit Mini-Diluter) with flow rate of 25 mL/min/tank (equals a complete volume replacement every 2.7 h) was used. The endpoints were hind limb length, body length (snout to vent [SVL]), developmental stage, wet body weight, thyroid histology, and daily mortality.

The corresponding mean measured concentrations in the definitive study were 5.97, 18.1, and 44.2 μg/L test item, respectively and thus within ±20 % of the nominal concentrations. No mortality was observed during the study. There were also no statistical significant differences between the median developmental stage of the test item treatments on SD 7 or at the conclusion of the study (SD 21) relative to the control. The occurrence of late stage specimens (>NF 60) was <20 % in the control and each treatment. Non-normalized and SVL-normalized HLL in the treatments were not significantly different from the control at SD 7 or SD 21. Asynchronous development was not noted in the control or test item treatments during the conduct of the study. SVL and body weight in the treatments were not significantly different from the control at SD 7 and at SD 21.

The prevalence of mild follicular cell hypertrophy and hyperplasia were consistent between control and test item-exposed X. laevis and treatment-related effects were not noted. The lack of treatment-related effects in this study is consistent with the absence of group-wise differences in median NF stage scores. No significant effects on behaviour or signs of overt toxicity were noted.

In conclusion, results from the present study indicated that X. laevis larvae exposed to the test item did not alter developmental rate based on developmental stage obtained at the conclusion of in-life exposure (SD 21). Using the decision criteria in the OECD 231, the test item did not affect amphibian metamorphosis or the thyroid axis directly at the concentrations tested based on the endpoints measured. Further, the test item did not affect larval growth (SVL and body weight) at the concentrations tested. To conclude, no endocrine disruptive effects were observed in this assay.

In the OECD 234 study freshly fertilized zebrafish eggs were exposed to the limit of solubility of the test substance under test conditions (48.6 µg/L) and dilution water control. A flow-through diluter system (Benoit Mini-Diluter) was used with flow rate to each tank of 2.7 L/h (6.5 volume exchanges/d). Exposure tanks were glass aquaria equipped with standpipes with an actual tank volume of 10 L. Thirty embryos per replicate tank, and 4 replicates per treatment (120 embryos/treatment) during exposure phases were used. Embryos from three clutches were double-selected using a dissecting microscope to ensure fertilization and normal appearing development and mixed prior to addition to replicate tanks. Exposure was started within 4 h after fertilization. The test chemical was delivered to the exposure chambers using a continuous-flow diluter. The exposure phase was conducted for 60 days post-hatch (dph). All exposure condition parameters were evaluated at pre-exposure phase d 0, in-life exposure phase d 0, and in-life test termination. The mean measured test item concentration was analytically determined to be 46.9 µg/L) which corresponds to +/- 20% of the limit solubility under test conditions. . The following parameters were determined throughout the exposure period - survival (daily), body weights and length, behaviour of hatchlings and juveniles (daily), vitellogenin (VTG), sex ratios, histopathology of the kidney, liver and gonads.

The target test item concentrations selected for the FSDT study were 0.0 (control) and 50 μg/L based on the practical solubility limit under test conditions of 48.6 µg/L determined prior to the start of the study in life phase. The corresponding mean measured concentration in the definitive study was 46.9 μg/L for the treatment and <LOD for the control. Mean survival in the control and the treatment group was 82.5 % and 81.7 %, respectively. The hatching success in the control and the treatment was 100 % and was complete by 4 days post-fertilization (dpf). Phenotypic sex ratio in the treatment was not significantly different from the control for both males and females. In addition, the incidence of undifferentiated phenotypic sex (undifferentiated gonad) was not significantly different from the control. Standard lengths and body weight in both female and male fish exposed to 46.9 μg/L test item were significantly less than those of the control group. Body weight and standard length in undifferentiated test item-exposed fish were not significantly different from the control, however, some size and weight reductions in exposed fish were already observed at this life stage. Plasma VTG levels in female and male fish exposed to the test item concentration were not significantly different from the control. Plasma VTG levels in undifferentiated fish exposed the test item were not significantly different from the control.

Exposure-related effects were observed in the liver including hepatocyte karyomegaly (minimal to moderate), oval cell proliferation (mild to moderate), single cell necrosis (minimal to mild), and increased mitotic figures (minimal). These findings were not distinct in undifferentiated or hermaphroditic fish. The liver findings of hepatocyte karyomegaly, oval cell proliferation, single cell necrosis (increased prevalence compared to controls) and increased mitotic figures in 46.9 μg/L test item-exposed fish were interpreted as indications of hepatic toxicity. Exposure-related effects in the gonads included decreased mean ovarian stage scores in 46.9 μg/L test item-exposed fish relative to controls. However, the delayed ovarian differentiation observed in the treatment-exposed females was related to the treatment-induced decrease in somatic growth. The seven instances of “intersex” gonads identified (four of which were identified in control fish) were not considered to be treatment-related. The subtle, but statistically insignificant, increase in the ratio of females to males in the test item-exposed fish compared to controls was most likely a function of delayed transition from the male to female phenotype as opposed to treatment-induced gonadal feminization. No significant effects on behaviour or signs of overt toxicity were noted.

In conclusion, the present study indicated that exposure to 46.9 μg/L test item (< 20% CV from the practical solubility under test conditions) did not reduce survival, alter sex ratio, increase the proportion of undifferentiated gonads, or alter plasma vitellogenin levels relative to control zebrafish. However, both standard length and body weight were reduced in female and male fish at the end of the exposure period (i.e. 60 dph). Early life stage exposure of zebrafish to the mean measured test concentration of 46.9 μg/L test item was associated with morphologic changes in the liver consistent with hepatotoxicity. It is reasonable to surmise that hepatotoxicity was responsible for the decreased somatic growth experienced by test item-treated fish, which in turn was likely the cause of delayed ovarian differentiation in females as determined by gonadal stage scoring. The low incidence of intersex in both control and test item-exposed fish, which manifested as either testicular oocytes, ovarian spermatogenesis, or hermaphroditism, was not considered to be related to the test item treatment, but was instead a function of gonadal immaturity in a species that is known to exhibit juvenile hermaphroditism. Based on these results, the NOEC was <46.9 μg/L and the LOEC was 46.9 μg/L. The highest tested concentration represented the limit of solubility under test conditions. However, it should be noted that the response observed was not related with an endocrine-mediated mode of action.

Based on the observations and results from the two conducted studies, it can be concluded that the test item does not possess endocrine disruptive properties to aquatic organisms. All observed effects in juvenile fish were not related to an endocrine-mediated mode of action.