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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 Nov 2020 to 18 Jan 2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

and OECD GD 23

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
-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
- Solubility in water: 0.104 mg/L (22°C, pH 7)

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. The highest stock solution was prepared in dimethylsulfoxide (DMSO; Merck, Darmstadt, Germany) at a concentration of 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, ranging from 0.0044 to 0.44 mg/mL, were prepared by dilution of the highest stock in DMSO. All stock solutions were clear and colourless after preparation.

- Final concentration: 0.44, 1.4, 4.4, 14, 44 and 140 µg/L.

Analytical monitoring:

yes

Details on sampling:

Samples for analysis were taken from all test concentrations and the controls.
One day before the start of exposure to check the functioning of the system and at the start, after 7, 8 , 9 , 14, and 21 days of exposure. In addition, samples were taken from the highest stock on day -2 (freshly prepared) and 0 (48 hours 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 highest stock solution on day -2, samples taken from the test solutions on day 8, and an additional sample taken from the solvent control on day 9 were stored in the freezer (≤-15°C) until analysis on day 0. 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)
-The highest stock solution was prepared in dimethylsulfoxide (DMSO) at a concentration of 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, ranging from 0.0044 to 0.44 mg/mL, 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): dimethylsulfoxide (DMSO)
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 µg/L (stock) and 10 µg/L (solvent control)

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Age at study initiation (mean and range, SD):To initiate the test, young daphnids < 24 hours old were selected.
- Stage and instar at study initiation: neonate daphnids
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
- Temperature of medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae (Chlorella sp.)
- Validity of the cultures: Historical data on the reproductive capacity are based on the numbers of living young counted three times a week in the individual cultures and tested to meet the validity criteria for survival and reproduction.
- Medium: M7
- Feeding during test: Continuous supply of Chlorella sp. (ca. 0.2 mg C/Daphnia/day)

ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not):
- Type and amount of food:
- Feeding frequency:
- Health during acclimation (any mortality observed): Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

196 – 250 (mg CaCO3)

Test temperature:

20 – 22 °C

pH:

7.5 – 8.1

Dissolved oxygen:

7.2 – 9.1 (mg O2/L)

Nominal and measured concentrations:

Nominal conc. (µg/L): 0.44, 1.4, 4.4, 14, 44 and 140.

Mean measured conc. (µg/L): 0.12, 0.96, 2.0, 5.5, 16 and 45

Details on test conditions:

TEST SYSTEM
- Test vessel: A stainless-steel vessel (~1.5 L) per test group, containing 4 mesh containers (i.e. replicates) of stainless-steel.
- Type (delete if not applicable): open
- Aeration: The test medium was prepared daily, constantly aerated.
- Test type: flow-through
- Renewal rate of test solution (frequency/flow rate): Continuously, with a flowrate of 700 mL/h.
- No. of organisms per vessel: 5 parental daphnids
- No. of vessels per concentration (replicates): 4 replicates per test group
- Medium: M7
- Light intensity: A daily photoperiod of 16 hours, with an intensity not exceeding 1000-1500 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Condition: Every day, the number of living, immobile and dead parental daphnids was recorded.
- Dead daphnids were removed when observed.
- Body length: At the end of the test.

Offspring
- Appearance first brood: When observed.
- Newborn daphnids: Every day, the number of newborn young was counted and the condition of the young recorded. Thereafter the young were removed.
- Presence of unhatched eggs: When observed.
- Incidence of immobility: When observed.
- Incidence of entrapment: When observed.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 45 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

Parental growth (body length)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

16 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

5.5 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

Survival and Condition of Parental Daphnids:
Two out of the twenty parental daphnids per group died during the test period in the solvent and blank control. Hence, parental mortality did not exceed 20% in the controls. As there was no difference in mortality between the blank and solvent control, data of these groups were pooled.
Mortality generally increased with increasing concentration of the test item, reaching 45% mortality at the highest concentration tested. Trend analysis revealed a significant trend in mortality, hence the effects on reproduction were calculated per introduced parental daphnid. Statistical analysis showed that mortality recorded at the two highest test concentrations was significantly different from the pooled control data.

Age at First Reproduction:
The first brood was observed at day 7 in all test groups and in all replicates of the different test concentrations. One exception was a single replicate of the solvent control in which the first offspring was recorded on day 8. All parental daphnids survived until appearance of the first brood, except for one specimen exposed to the highest test concentration which died on day 1. Based on the above, it can be concluded that the test item did not cause delay in onset of reproduction at the range of concentrations tested.

Reproduction:
On average, 132 and 117 offspring were produced per introduced daphnid in the blank and solvent controls, respectively. Statistical comparison revealed that there was no significant difference between the blank and solvent control, and therefore, data of these groups were pooled.
The number of offspring was comparable to the pooled control in the four lowest test concentrations, while reduction of 4.7 and 28% was found at mean measured concentrations of 16 and 45 µg/L, respectively. Statistical analysis showed that the reproduction of the daphnids was significantly reduced at the highest test concentration.

The numbers of aborted eggs observed throughout the test were considered not relevant, while considerable numbers of immobile young were recorded at all test concentrations. Offspring immobility seems to be induced by exposure to the test item as only three immobile young were observed in the control groups. However, offspring immobility did not follow a clear dose-response pattern and the main response variable ‘number of living offspring per introduced parent’ was not significantly affected at the five lowest test groups. The latter indicates that, although immobile offspring was produced, the production of living offspring was not hampered at mean measured concentrations up to and including 16 µg/L which is considered the most biologically relevant conclusion. It should also be noted that majority of the immobile offspring were recorded in a single event (day 14). Considering the above, no conclusion can be drawn on the actual cause for the immobile offspring. Due to lack of dose-relation, statistical analysis was not performed.

Parental Body Length:
Statistical comparison revealed that there was no significant difference between the blank and solvent control, and therefore, data of these groups were pooled.
No statistically significant reduction in body length was found at the range of concentrations tested. The NOEC was at the highest test concentration, while the LOEC was beyond the range tested.

Reported statistics and error estimates:

Parental mortality:
• Trend analysis: Cochran Armitage Test (one-sided greater, alpha = 0.05)
• NOEC: Step-down Cochran-Armitage Test (one-sided greater, alpha = 0.05), preceded by a qualitative trend analysis by contrasts (monotonicity of the dose-response curve).
• LCx values were calculated with Probit analysis using linear max. likelihood regression with probits of the percentages of mortality at the end of the test as function of the logarithms of the corresponding concentrations. It should be noted that the lowest concentration was excluded from the analysis as it deviated from the dose-response and prevented good fit of a model.

Parental body length:
• Data distribution: Shapiro-Wilk´s Test
• Homogeneity of variance: Levene´s Test (with Residuals)
• NOEC: Williams Multiple Sequential t-test (one-sided smaller, alpha = 0.05) preceded by a trend analysis by contrasts (monotonicity of the dose-response curve).
• ECx values for body length could not be calculated since the recorded effects were below 10%.

Reproduction:
• Data distribution: Shapiro-Wilk´s Test
• Homogeneity of variance: Levene´s Test (with Residuals)
• NOEC: Williams Multiple Sequential t-test (one-sided smaller, alpha = 0.05) preceded by a trend analysis by contrasts (monotonicity of the dose-response curve).
• ECx-values: The ECx values were determined using the Logit analysis using maximum likelihood regression with the logits of the percentages of cumulative reproduction of the three highest groups at the end of the test as function of the logarithms of the corresponding concentrations. The three lowest concentrations were not included in the analysis as they showed a stimulation, rather than inhibition of reproduction preventing proper model fitting. It should be noted that replicate instead of group means were used in this model. The 95%-confidence intervals were obtained by bootstrapping (1,000 re-samplings).

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate] did not affect survival of Daphnia magna at mean measured concentrations up to 5.5 µg/L after 21 days of exposure (NOEC). Reproduction was not affected at mean measured concentrations up to 16 µg/L (NOEC).
Exposure to a mean measured concentration of 45 µg/L induced significant inhibition of the reproductive capacity of the parental daphnids (LOEC). Survival of parental daphnids was significantly affected at mean measured concentrations of 16 µg/L (LOEC) and higher.
No significant effects on growth and age at first reproduction of parental daphnids was found at the range of concentrations tested. The NOEC for these endpoints was >45 µg/L.

Description of key information

There is one GLP guideline study according to OECD 211 and OECD 23 available on the long-term toxicity to invertebrates in a flow-through system. To investigate the reproductive capacity output of Daphnia magna, test organisms (young female daphnids, aged less than 24 hours at the start of the test) were exposed for 21 days to aqueous solutions containing the test item at various concentrations. 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, the highest stock solution was prepared in dimethylsulfoxide (DMSO; Merck, Darmstadt, Germany) at a concentration of 1.4 mg/mL.

Under the conditions of the present key study Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate] did not affect survival of Daphnia magna at mean measured concentrations up to 5.5 µg/L after 21 days of exposure (NOEC). Reproduction was not affected at mean measured concentrations up to 16 µg/L (NOEC).

Exposure to a mean measured concentration of 45 µg/L induced significant inhibition of the reproductive capacity of the parental daphnids (LOEC). Survival of parental daphnids was significantly affected at mean measured concentrations of 16 µg/L (LOEC) and higher.

No significant effects on growth and age at first reproduction of parental daphnids was found at the range of concentrations tested. The NOEC for these endpoints was >45 µg/L.