2-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol

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:

2013-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The test substance is insoluble in water (information from sponsor). Therefore the test solution was prepared following general guidance provided in OECD 23. The test solution was prepared by directly adding test substance to test medium according to the loading rate described in the table below. The mixture was ultrasonicated for 15 minutes at 40°C. Then the test solution was stirred on a magnetic stir plate for approx. 24 hours °C.

Prior to toxicity testing, each solution was filtered (Schott glass filtration unit with 0.2 μm nylon membrane, Whatman) to assure the removal of any undissolved test substance. Filters were first wetted and rinsed with at least 100 mL of highly distilled water (Milipore water). Once wet, filters were not allowed to dry out, so some residual water was left standing over the filter. Approximately 800 mL of the test solution was then added to the filtration unit and the first 50 – 90 mL were discarded in order to saturate the filter. The remainder of the test solution was bulk filtered to the point where approximately 10 mL remained standing over the filter. The filtered test solution was used for toxicity testing. Control test media was similarly filtered.

Undissolved test substance/particulate material was visible in the whole test solution. After filtration, all test solutions were visibly colorless and clear throughout each renewal period.

The concentration of the stock solution was 10 mg/L.

Analytical methods for the quantitative determination of the test substance dissolved in aqueous test media were investigated in previous aquatic toxicity and physical chemistry studies. The dissolved concentrations of the test substance were below analytical detection limits (0.10 mg/L in acute Daphnia study, information from sponsor) and could not be confirmed.

Due to the low water solubility of the test substance, the stability of the test substance as a solution in test media and under testing conditions could not be evaluated. The test solutions were visibly homogeneous.

Analyses of the test substance preparations were not conducted, since a reliable method for analyses in the required concentration range was not provided.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test species : Daphnia magna STRAUS
Reason for selection of the test species: Recommended species in the test guidelines
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

Reference substance testing: In order to verify that the Daphnia magna culture is responding normally to toxic stress, tests with a reference substance are conducted monthly. Reference substance tests are conducted generally according to the OECD 202 guideline and in accordance with GLP, but without a GLP status. The EC50(48 h) of the reference substance sodium chloride (NaCl) was 5.13 g/L (experiment date: 04 Sep 2013).
This result is within the range of 3.88 – 7.22 g/L, which represents ±2 standard deviations from the published EC50(48h) of 5.55 g/L and indicates that the culture of Daphnia magna used in this study is responding normally to toxic stress.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

2.39 - 2.43 mmol/L

Test temperature:

20 - 21 ºC

pH:

7.7 - 8.2

Dissolved oxygen:

4.3 - 4.7 in the freshly prepared test solutions
8.3 - 8.8 before renewal in the old solutions

Salinity:

n/a

Nominal and measured concentrations:

nominal 10 mg/L

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: 687 - 791 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: Semi-static, renewal 3 times per week
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 1630 mg TOC/L, 1690 mg TOC/L and 1870 mg TOC/L (respectively) in the algal concentrates used. The daphnids were fed a defined volume (≤123 μL) of the concentrate to reach the amount of food defined in the table below. 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 72 h test interval with 3 feedings, 0.123 mL each, a total of 0.369 mL are added to 50 mL test volume resulting in a maximal dilution of 0.7%.

Reference substance (positive control):

yes

Remarks:

sodium chloride

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

No significant mortality or reduced reproduction were observedin any of the test treatments. A statistically significant effect on growth was observed at 10 mg/L; however there was no effect on reproduction in this test group so the slight reduction in growth is not considered toxicologically significant. No 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.

The concentration of test substance in test media was not verified analytically. According to guidance in 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.

Validity criteria fulfilled:

yes

Conclusions:

No significant mortality or reduced reproduction were observed in any of the test treatments. A statistically significant effect on growth was observed at 10 mg/L; however there was no effect on reproduction in this test group so the slight reduction in growth is not considered toxicologically significant. No 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.

Description of key information

Based on long-term (chronic) toxicity study data, the product is very likely not harmful to aquatic invertebrates. 

Additional information

To assess the toxic potential of the substance a chronic toxicity study according to GLP guideline 211 and under consideration of GLP was conducted (BASF 2014). The test was conducted as limit test with a nominal loading rate of 10 mg/L. Concentration control analysis was not performed because a reliable method for analyses in the required concentration range could not be developed. However, all reasonable efforts were taken to produce a saturated solution of the test substance in test media, following the guidance in OECD 23 and the test solutions were freshly prepared and exchanged 3 times per week to ensure consistent exposure conditions for the duration of the test. No significant mortality, reduced reproduction or any other additional significant adverse effects or abnormal behavior were observed in any of the test treatments. The LOEC was determined as > 10 mg/L and the NOEC as >= 10 mg/L.