Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate]

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 Oct 2020 to 26 Nov 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: 0021426801
- Purity, including information on contaminants, isomers, etc.: 99.3%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was not sufficiently soluble in water to allow preparation of a stock solution that could be dosed in a flow-through system. Therefore, an organic solvent was used to prepare stock solutions. Stock solutions were prepared in dimethylsulfoxide (DMSO; Merck, Darmstadt, Germany) at concentrations ranging from 0.060 to 1.4 mg/mL. No other treatment than a vigorous shaking was needed to obtain compete dissolution of the test item in the solvent. Lower stock concentrations were prepared by dilution of the highest stock in DMSO. All stock solutions were clear and colourless after preparation.

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the controls.
- Frequency: One day before the start of exposure to check the functioning of the system and at the start, after 7, 14, 21 and 28 days of exposure. In addition, samples were taken from the highest stock on day -1 (24 hours old) and 0 (48 hours old) after preparation to check the stability of the stocks under test conditions. All stock solutions containing test item were sampled on day 7.
- Volume: 1.75 mL
- Sample storage conditions before analysis: Samples taken from the stock solution(s) on day -1 and 7 were stored in the freezer (≤-15°C) until analysis. All other samples were transferred to the analytical laboratory at the Test Facility for fresh analysis and were analysed on the day of sampling.

Vehicle:

yes

Remarks:

dimethylsulfoxide (DMSO; Merck, Darmstadt, Germany)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Stock solutions were prepared in dimethylsulfoxide (DMSO) at concentrations ranging from 0.060 to 1.4 mg/mL. No other treatment than a vigorous shaking was needed to obtain compete dissolution of the test item in the solvent. Lower stock concentrations were prepared by dilution of the highest stock in DMSO. All stock solutions were clear and colourless after preparation. The stock solutions were dosed to test medium by means of a computer-controlled dosing system. Stock solutions were renewed three times a week.
- Controls: Blank and solvent control
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): DMSO

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
Common name: Fathead minnow (Pimephales promelas, Teleostei Cyprinidae) Rafinesque
Source: In house culture.
Holding medium: Test medium

Measurements
Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week
Temperature: continuous
In addition, temperature was measured before transferring the parental fish to the breeding system.
Water quality parameters: Will be kept within the optimum limits for the respective fish species.
Ratio male/female: 1:2
Spawning tank: The spawning tank is equipped with a substrate (pvc-tube), which enables collection of the fertilised eggs.
Feeding brood stock: Frozen brine shrimp Nauplii and pelleted fish food (SDS 400, Coppens International bv, Helmond, The Netherlands).
Time of fertilisation: Males and females are put together in spawning tanks and spawning starts the following day approximately 1 to 2 hours after lights have been switched on.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Hardness:

214-232 (mg CaCO3)

Test temperature:

23-25 °C

pH:

7.2 - 7.7

Dissolved oxygen:

6.6 - 9.7 (mg O2/L)

Nominal and measured concentrations:

Nominal conc. (µg/L): 6.0, 13, 29, 64 and 140.
Mean measured conc. (µg/L): 4.0, 8.8, 20, 44, and 115

Details on test conditions:

Test type: Flow-through, with continuous renewal of test media
Test duration: 32 days
Introduction egg: before cleavage of the blastodisc commenced (approximately 2-4 hours after fertilisation)
Test vessels: Stainless steel vessels (~1.7 L).
Experimental design: The experiment started with 80 fresh and healthy fertilised fathead minnow eggs per test group (nominal day 0). The embryos were randomly distributed and divided equally over 4 stainless-steel vessels.
Light period: A daily photoperiod of 16 hours.

Feeding
Embryonic phase: no feeding.
Larvae and juvenile fish: Brine shrimp Nauplii 24 or 48-hours old. Food was supplied ad libitum.

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

> 115 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Embryonic survival (day 7)

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

> 115 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

Post-hatch survival (day 32)

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

20 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Remarks:

Larval growth (body length)

Key result

Duration:

32 d

Dose descriptor:

NOEC

Effect conc.:

8.8 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

Larval growth (body weight)

Details on results:

Embryonic Survival
Hatching started on day 4 in majority of the replicates of the controls and three lowest test concentrations. At a mean measured concentration of 44 µg/L, the first larvae appeared on day 4 in a single replicate, while hatching started on day 5 in the other replicates. Hatching started on day 5 in all replicates of the highest test concentration. Hatching was complete on day 7 in all test groups. Hence, there was a slight delay in onset of hatching at mean measured concentrations of 44 µg/L and higher, yet not significant.
The overall survival of embryos at the end of hatching was 93 and 95% in the blank and solvent control, respectively. Hence, the validity criterion of >70% survival until completion of hatching was met. Statistical comparison revealed that there was no significant difference between the blank and solvent control, and therefore, data of these groups were pooled.
Hatching success was between 95 and 98% at all test concentrations. Since hatching success was higher at any concentration compared to the pooled control, no statistical analysis was needed to determine the NOEC. The NOEC for hatchability was 115 µg/L, while the LOEC was beyond the range of concentrations tested.

Post-hatch Survival and Development
The mean post-hatch larval survival was 93% for the blank control and 87% for the solvent control. Hence, the validity criterion for post-hatch survival of at least 75% was met. Statistical comparison revealed that there was no significant difference between the blank and solvent control, and therefore, data of these groups were pooled.
Survival rates varied between 86 and 92% at the different test item treatments. Recorded mortality was not concentration-dependent. Statistical analyses confirmed that larval survival was not significantly affected at any of the concentrations tested. The NOEC for post-hatch survival is 115 µg/L.
Few clinical effects were recorded throughout the test. Most prominent symptoms included malformations of the body, but the severity and incidence of these symptoms was not concentration-dependent.

Effects on Larval Growth
Statistical comparison revealed that there was no significant difference between the blank and solvent control for both body weight and length, and therefore, data of these groups were pooled.
Statistically significant weight reduction was found at mean measured concentrations of 20 µg/L and higher. Reduction in body weight was comparable at the three highest concentrations tested, i.e. was ~10% at mean measured concentrations of 20 µg/L and higher. It was not possible to estimate an EC10 for body weight reduction using regression analysis due to poor model fit. The EC10 for body weight was set at 20 µg/L, i.e. the lowest test concentration that induced 10% reduction. Consequently, a 95%-confidence interval could not be determined for this threshold concentration.
Body length reduction increased with increasing test item concentration, reaching 3.6% reduction at a mean measured concentration of 115 µg/L. A statistically significant reduction was found at the concentrations of 44 and 115 µg/L.
It should be noted that two larvae exposed to the highest test concentration and one larva exposed to the second highest concentration were observed to be considerably smaller compared to the other specimens in the respective replicates. Pictures of these fish are presented in Appendix 4. Since the deviant larva exposed to a mean measured concentration of 44 µg/L (target concentration of 64 µg/L) had severe scoliosis, it was not possible to measure its total length reliably. Therefore, this fish was not included in the statistical analysis for body length.

Reported statistics and error estimates:

Comparison of control treatments:
• Hatchability and post-hatch survival: Fisher`s Exact Binomial Test (alpha=0.05, two-sided)
• Body weight and length: Student t-test for Homogeneous Variances (alpha=0.05, two-sided) after check for normality of distribution and homogeneity of variances.
Since no statistically significant differences were detected for all parameters, data of the control groups were pooled for each endpoint.

Hatchability (embryonic survival):
• ECx values could not be calculated as ≥95% embryos hatched in all treatments.
• No statistical analysis was needed to determine the NOEC, since hatching success was higher at every concentration compared to the pooled control.

Post-hatch survival:
• ECx values could not be calculated for several reasons: post-hatch mortality was 10% in the control, whereas in test concentrations between 7.8 am 14%. Moreover, the observed mortality was not dose-related.
• Differences between treatments and the pooled control: Chi²-2 x 2 table test with Bonferroni correction (α=0.05, one-sided greater) preceded by a qualitative trend analysis by contrasts (monotonicity of concentration/response).

Larval growth (body length and body weight):
• ECx values for body length could not be calculated since the recorded effects were below 10%. The ECx values for body weight could not be estimated, due to absence of a dose-dependent effect at the range of concentrations tested, as regression analysis resulted in a poor model fit. Alternatively, the EC10 was set at the lowest test concentration that induced 10% body weight reduction. Consequently, a 95%-confidence interval could not be determined.
• Differences between treatments and the controls:
• Data distribution: Shapiro-Wilk´s Test
• Homogeneity of variance: Levene´s Test (with Residuals)
• Comparison with the controls: Williams MultipleSequential t-test Procedure (α=0.05, one-sided smaller) after trend analysis by contrasts (monotonicity of concentration/response).

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the present study assessed the possible lethal and sub-lethal effects of Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate] during the embryonic and early larval development of the fathead minnow (Pimephales promelas). The results led to the following conclusions for:

1. Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate] did not affect the hatching success (embryonic survival) at mean measured concentrations up to and including 115 µg/L (NOEC). Hence the LOEC was beyond the maximum concentration tested.

2. Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate] did not affect post-hatch survival at mean measured concentrations up to and including 115 µg/L (NOEC). The LOEC was beyond the maximum concentration tested.

3. Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate] had a significant effect on the growth of the exposed larvae. Body weight was significantly affected at mean measured concentrations of 20 µg/L (LOEC) and higher, while body length reduction was statistically significant at concentrations of 44 µg/L (LOEC) and the highest concentration tested. The NOEC for body weight was 8.8 µg/L, while the NOEC for body length was 20 µg/L.

Description of key information

There is one GLP guideline study according to OECD 210 and OECD 23 available on the long-term toxicity to fish in a flow-through system. Early-life stages of fathead minnow (Pimephales promelas) were exposed to a range of concentrations of the test item dissolved in water until 32 days post-fertilization. The test item was not sufficiently soluble in water to allow preparation of a stock solution that could be dosed in a flow-through system. Therefore, stock solutions were prepared in dimethylsulfoxide (DMSO; Merck, Darmstadt, Germany) at concentrations ranging from 0.060 to 1.4 mg/mL.

The following results were obtained in this study:

1. Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate] did not affect the hatching success (embryonic survival) at mean measured concentrations up to and including 115 µg/L (NOEC). Hence the LOEC was beyond the maximum concentration tested.
   
   


2. Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate] did not affect post-hatch survival at mean measured concentrations up to and including 115 µg/L (NOEC). The LOEC was beyond the maximum concentration tested.
   
   


3. Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate] had a significant effect on the growth of the exposed larvae. Body weight was significantly affected at mean measured concentrations of 20 µg/L (LOEC) and higher, while body length reduction was statistically significant at concentrations of 44 µg/L (LOEC) and the highest concentration tested. The NOEC for body weight was 8.8 µg/L, while the NOEC for body length was 20 µg/L.