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Long-term toxicity to aquatic invertebrates

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long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD guideline and performed under GLP.
according to guideline
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on test solutions:
The test substance is poorly soluble in water. Therefore the test solutions were prepared following general guidance provided in OECD 23 in order to generate a saturated solution of the test substance in test medium. Generally, the test solution is prepared by directly adding test substance to test medium according to the loading concentration. The test solution was prepared by directly adding 20 mg of test substance in 2L of test medium. The mixture was placed in an ultrasonic bath for 15 minutes then stirred for about 1 day. Undissolved test substance was visible at the water surface and in the stock solution. Undissolved test substance was removed by filtration with a membrane filter (pore width 0.2 μm, Celluloseacetat, Whatman OE66). Fresh test solutions were prepared daily. All stock solutions were visibly clear after filtration.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Test species: Daphnia magna STRAUS
- Origin: The clone of Daphnia magna STRAUS 1820 used was supplied by the Institut National de Recherche Chimique Appliquée, France, in 1978.
From this date on this clone was cultured and bred continuously in the Ecotoxicology Laboratory of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen Germany.
- Culture conditions: Daphnia brood stock are kept in mass cultures consisting of approx. 20 – 30 individuals for a maximum of 4 weeks. All
individuals in the mass culture originate from a single female. After approximately 14 days the adults have produced at least 3 broods and the young can be used in tests. Offspring are removed from the mass cultures at least once daily during the normal work week to ensure that young daphnia are <24-h old (first instar) at test initiation. Detailed records are kept (in test facility archives) to monitor the health of Daphnia brood stock cultures including observations of young production, mortality, ephippia, and measurement of water chemistry parameters.
Only young from healthy cultures without signs of stress are used for testing.
- Acclimatization: The Daphnia are cultured under the identical conditions as the test including test media (Elendt M4), water quality, temperature (20 ±1°C), and diet.
- Age at test initiation: < 24 h.
Test type:
Water media type:
Limit test:
Total exposure duration:
21 d
Total Hardness: 2.41 – 2.46 mmol/L
Test temperature:
20 °C
7.9 - 8.1
Dissolved oxygen:
4.4 - 8.8 mg/L
Nominal and measured concentrations:
- Nominal concentrations: 0 (control) and 10 mg/L.
- Measured concetrations: not performed.
Details on test conditions:
- Test medium: A synthetic fresh water (Elendt M4) is used as media for culture and test purposes. For the composition of this M4 medium see OECD 211. The general properties of this medium are as follows: Total hardness: 2.20 – 3.20 mmol/L; Acid capacity up to pH 4.3: 0.80 – 1.00 mmol/L; Molar ratio Ca:Mg: about 4 : 1; pH value: 7.5 – 8.5; Conductivity: 550 - 650 μS/cm; Total organic carbon: < 2 mg/L; Dissolved oxygen: Must remain ≥ 3mg/L during the test. To assure optimal dissolved oxygen levels, the M4 medium is aerated for approximately 24 h prior to use.
- Test vessels : Numbered glass beakers (nominal volume 100 mL), covered with glass Petri plates to slow evaporation.
- Test volume: 50 mL
- Biological loading: 1 animal / test vessel (0.02 animals / mL)
- Light intensity / Photo period: 648-679 lux at a wave length of 400-750 nm (recommended values in lux units provided in ISO 10706); 16 h light : 8 h darkness
- Aeration: none
- Test solution renewal: Due to the instability of the test substance in the test medium, this study was conducted as a static-renewal exposure
- Diet: During the test daphnids were fed daily a diet of live green algae Desmodesmus subspicatus, cultured in a synthetic medium. The algae were separated from their culture medium by centrifugation, resuspended in daphnid's medium (M4) corresponding to concentrations of 1770 mg and 1960 mg TOC*/L (respectively) in the algal concentrates used. The daphnids were fed a defined volume (≤113 μL) of the concentrate to reach the amount of food required according to guideline. The algae were stored in a refrigerator (dark, about 4-8°C) for maximum 21 days. By adding the algal concentrate the test solution was slightly diluted. During a 24 h test interval with 1 feeding, 0.113 mL are added to 50 mL test volume resulting in a maximal dilution of 0.2%.

Each test group initially consisted of 10 replicates containing one juvenile Daphnia (<24 h old) each. The concentration for this study was selected based on the result of an acute Daphnia study. In this acute study no immobilisation was observed at a loading rate of 100 mg/L. According to OECD-guideline, the highest suggested test concentration is 10 mg/L for a limit test.

The test was initiated by impartially distributing 1 neonate (<24h old) Daphnia magna into each of the 10 replicate test vessels per test group. The test vessels were maintained and daphnids fed.
Parent mortality, abnormal effects, and numbers of live and dead offspring were assessed every day throughout the experiment. Reproductive success was measured by counting and discarding the offspring produced by each parent for the duration of the study. Separating neonates from adults was accomplished by gently removing adult daphnids from each chamber by means of a wide bore pipet and pouring remaining water and young daphnids through a fine mesh screen. The young collected on the screen were placed in a petri plate and counted on a light table before being discarded. On each day of the test the parent daphnids were returned to their test vessels with fresh test solution.
On day 21 of exposure, the surviving adults were removed from the test vessels and euthanized by immersion in 70% isopropanol. Individuals were then isolated on a labeled glass slide. The body length of each adult daphnid was measured from the apex of the helmet to the base of the posterior spine (i.e. excluding the anal spine and protruding second antennae) with a binocular dissecting microscope (Leitz Diavert) and a calibrated eyepiece micrometer which was checked with a standardized slide.
Throughout the test, the appearance of the test solutions and dissolution behavior of the test substance was observed and recorded daily. The chemical and physical parameters of the test medium (total hardness, acid capacity, pH, conductivity and total organic carbon) were determined after aeration and prior to use in the test and were within acceptable ranges. Dissolved oxygen, pH and temperature were measured in the old and in the freshly prepared test solution in replicate 1 of each test group for one interval per week. In addition, temperature was measured continuously during the whole exposure period in a separate vessel filled with water proximal to the test vessels. Hardness was measured in the freshly prepared test solution in an additional replicate of each concentration for one interval per week and in the old test solution from the combined replicates of each test group for one interval per week.
Reference substance (positive control):
Sodium Chloride
21 d
Dose descriptor:
Effect conc.:
>= 10 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: No effects were seen below the LOQ
21 d
Dose descriptor:
Effect conc.:
> 10 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
Remarks on result:
other: No effects were seen below the LOQ
Details on results:
One mortality was observed in the 10 mg/L test group (10% mortality). This minor level of effect is within the accepted tolerance for control performance (OECD 211) and was thus not toxicologically significant. No significant mortality or any other additional significant adverse effects or abnormal
behavior were observed in any of the test treatments. The data were not sufficient to calculate ECx values for reproduction or mortality.

All test solutions were visibly colorless and clear throughout each renewal period. No undissolved test substance or other unusual manifestations were observed.

In the other biological observations a very slight change in all parameters was observed at the test concentration compared to nominal, however this change is considered too small to be considered significant.
Results with reference substance (positive control):
The EC50(48 h) of the reference substance sodium chloride (NaCl) was 4.63 g/L (experiment date: 09 Oct 2013). This result is within the range of 3.88 – 7.22 g/L, which represents ±2 standard deviations from the published 48-h EC50 of 5.55 g/L and indicates that the culture of Daphnia magna used in this study is responding normally to toxic stress.
Reported statistics and error estimates:
Statistical evaluations were performed to determine LOEC and NOEC. The following parameters and statistical procedures were used to evaluate significant differences to the control:
- Reproduction as number of living young (Wilcoxon’s test, one sided analysis)
- Growth as length in mm (Students t-test, one sided analysis)
- Time to first brood (Wilcoxon’s test, one sided analysis)
- % immobile young (Wilcoxon’s test, one sided analysis)
- % Aborted eggs (Wilcoxon’s test, one sided analysis)

The results of the statistical analyses were used to aid in the determination of the NOEC and LOEC. However, scientific judgment was used to determine if statistical differences were biologically meaningful. In this limit test with one test concentration, the NOEC was defined as greater than or equal to the saturation concentration under test conditions when no significant treatment-related adverse effects on survival, reproduction or growth are observed. The LOEC was defined as greater than the NOEC when no significant treatmentrelated effects on survival, reproduction or growth are observed. Effect concentrations (ECx)
could not be calculated since there were no effects at the tested concentrations.

Mortality and reproduction summary after 21 days exposure

Nominal test concentrations (mg/L)



Mean living per surviving adult

% effect

Parent animals

% effect

0 (control)










Other observations among surviving parent animals after 21 days exposure

Nominal test concentrations (mg/L)

Mean Growth (length, mm)

% immobile young

Mean days to first brood

% aborted eggs

0 (control)










Validity criteria fulfilled:

Description of key information

The 21-d NOEC is ≥10 mg/L in aquatic invertebrates (Daphnia magna).

Key value for chemical safety assessment

Additional information

In a 21-day chronic toxicity study, Daphnia magna were exposed to Thiodiethylene bis[3- (3,5-di-tert-butyl-4-hydroxyphenyl)propionate] at nominal (loading) concentrations of 0 (control) and 10 mg/L under semi-static, daily renewal conditions in accordance with the OECD 211 guideline.

The water pH, temperature and dissolved oxygen were within acceptable guideline specifications. Mortality, reproduction and sublethal effects were observed daily. The following overall effect concentrations (mg/L) are for the most sensitive endpoint (reproduction) and were obtained based on nominal (loading) concentrations: LOEC > 10 mg/L NOEC ≥ 10 mg/L. No significant mortality or any other additional significant adverse effects or abnormal behavior were observed in any of the test treatments. The data were not sufficient to calculate ECx values for reproduction or mortality.

Concentration control analysis was not performed since the analytical detection limit was above the water solubility of the test substance. However, all reasonable efforts were taken to produce a saturated solution of the test substance in test media, following the guidance in OECD 23. According to OECD 23, for tests with chemicals that cannot be quantified by analytical methods at the concentrations causing effects, the effect concentration can be expressed based on the nominal concentrations. The results in this study are consistent with all validity criteria and the test is valid according to the guidelines of this study. No deviations from test guidelines or other incidents occurred during the course of the reported test which may have influenced the results.

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