A mixture of: isomers of 2-(2H-benzotriazol-2-yl)-4-methyl-(n)-dodecylphenol; isomers of 2-(2H-benzotriazol-2-yl)-4-methyl-(n)-tetracosylphenol; isomers of 2-(2H-benzotriazol-2-yl)-4-methyl-5,6-didodecyl-phenol. n=5 or 6

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:

10.08. - 01.12.2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study, GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

adopted on September 21, 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
For the determination of the test item concentrations, duplicate samples were taken from the freshly prepared test media of all test concentrations and the solvent control at the start of the test (Day 0), and thereafter at every second test medium renewal (Days 5, 10, 14, 19). To determine the maintenance of the test item concentrations during the test medium renewal periods, two stability controls were performed (Day 0 to Day 3 and Day 10 to Day 12), corresponding to the two different renewal periods of 72 and 48 hours. The following stability samples were taken in duplicate:
a) Samples with food, incubated during the renewal periods under the same conditions as the test itself (however without daphnids). Sampling from the test itself was not possible, since the test medium volumes were too small for the analytical requirements.
b) Samples without food and test animals, incubated during the renewal periods under the same conditions as the test itself.
- Sample storage conditions before analysis: All samples were deep-frozen (at about -20 °C) immediately after sampling.

The concentrations of the test item were analyzed in at least one of the duplicate test medium samples from the highest nominal concentration of 0.20 µg/L, determined in the experiment as the NOEC. From the solvent control samples only one of the duplicate samples was analyzed from the sampling dates at Day 0, 3, 10, 12, and 19. The test concentrations below the NOEC were not analyzed, because they were not relevant for the interpretation of the biological results.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
Prior to the start of the test and prior to each test medium renewal, the test media and the
controls were prepared as follows:
First, a concentrated solution of the test item in DMF with a nominal concentration of 2.0 mg/mL was prepared by dissolving nominal 40 mg (range: 38.7 - 40.4 mg) of the test item completely in 20 mL DMF. In a series of subsequent dilution steps this concentrated solution was diluted with DMF to prepare the application solutions used for the dosage of the test media with different test concentrations. Then, identical volumes of each of the application solutions were dosed into test water to prepare test media of the corresponding test concentrations. For the preparation of the solvent control, DMF without test item was dosed into test water. The concentration of DMF was the same (100 pL DMF per liter test water) in all test media and in the solvent control.
- Controls: test water without addition of test item or solvent was used.
- Chemical name of vehicle: N,N-Dimethylformamide (DMF)
- Concentration of vehicle in test medium: 100 µL DMF/ L test water

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia Magna
- Strain/clone: clone 5
- Source: in-house breed
- Age of parental stock: The daphnids used for the test originated from parental daphnids that were at least 14 days old but not older than four weeks and were not first brood progeny. At the start of the test, the test animals were less than 24 hours old.
- Feeding during test: yes
- Food type: with green algae of the species Scenedesmus subspicatus and with a fish food suspension.
- Amount: not specified
- Frequency: three times a week

ACCLIMATION
- Acclimation period: no

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

2.5 mmol/L (= 250 µg/L as CaCO3)

Test temperature:

19-20 °C during the test period

pH:

between 7.7 and 8.0 during the test.

Dissolved oxygen:

between 8.2 and 9.2 during the test.

Nominal and measured concentrations:

Nominal: 0.013, 0.025, 0.050, 0.10, and 0.20 µg/L
0.20 µg/L: Mean measured concentration: 0.09 µg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL beaker
- Type: test vessels were covered with glass plates
- Material, size, headspace, fill volume: glass beaker containing 80 mL of test medium.
- Aeration: Before use, the test water was aerated until oxygen saturation. During the test, the test media were not aerated.
- Renewal rate of test solution (frequency/flow rate):
In this semi-static test, the test media of all test concentrations and of the control were renewed on Days 3, 5, 7, 10, 12, 14, 17, and 19 of the test period (every Monday, Wednesday, and Friday). In the end, a total of 8 test medium renewals were performed.
- No. of organisms per vessel: 1 (kept individually)
- No. of vessels per concentration (replicates): 10
- No. of vessels per vehicle control (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water
- Culture medium different from test medium: No
- Intervals of water quality measurement:
At the beginning and end of each test medium renewal period, the pH values and dissolved oxygen concentrations were measured in one of the beakers of each test concentration and of the control and of the solvent control. At the same time periods, the water temperature was measured in one of the control beakers. Additionally, the air-temperature in the testing room was continuously recorded by a temperature recorder. The appearance of the test media was visually recorded at each of the above mentioned intervals.

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: Photoperiod of 16 hours light and 8 hours darkness, with a 30 minute transition period (light intensity during light period within the range of about 470 and 640 Lux)
- Light intensity: 470 - 640 LUX

EFFECT PARAMETERS MEASURED:
The test replicates were observed for mortality of adults at the start of the test, on the first and third day after treatment and thereafter three times per week before renewal of test media. On the same observation dates, starting from Day 5, the number of offspring was recorded. Dead animals were removed at the renewal of the test media. The reproduction rate was calculated as the total number of living offspring produced per parent female surviving until the end of the test.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study:
The test concentrations were based on the results of a range finding test and on preexperiments to the dosage of the test item in test water (without GLP). Concentrations far above the water solubility limit of the test item in test water (0.15 pg/L) were not tested.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.2 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.09 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.2 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.09 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Details on results:

- Mortality of parent animals: At the end of the test, the survival rate of the test animals was 90% in the control and 100% in the solvent control and at all test concentrations
- No. of offspring produced per day per female: The mean reproduction rate of the daphnids In the solvent control was 94.5 ± 8.0 living offspring per
adult (mean ± SD). The corresponding value in the control was 97.9 ± 4.4.
- Body length and weight of parent animals: no data
- Type and number of morphological abnormalities: not observed
- Type and number of behavioural abnormalities: not observed
- Number of males and females (parental): not specified

Reported statistics and error estimates:

The NOEC and the LOEC of the reproduction rate were statistically evaluated by testing the mean reproduction rate at the test concentrations for statistically significant differences to the solvent control value by the multiple Williams-test after a one-way analysis of variance (ANOVA). The EC50 of the reproduction rate after 21 days could not be calculated due to the absence of a toxic effect of the test item on the reproduction rate up to the highest test concentration.

TEST ITEM CONCENTRATION

The measured test item concentration in the analyzed test medium of nominal 0.20 µg/L varied in the range of 22 to 87% of the nominal value at the start of the test medium renewal periods. At the end of the test medium renewal periods of two and three days, the measured concentration in the samples with and without food was in the range of 17 to 66% of nominal.. Using the measured concentrations, the mean measured concentration (calculated as the average over all measurements) was 0.09 µg/L (45% of nominal).

Description of key information

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

Additional information

The effect of the test item on the survival and reproduction of Daphnia magna was investigated in a semi-static test over 21 days according to the OECD Guideline 211. The nominal test concentrations were 0.013, 0.025, 0.050, 0.10, and 0.20 µg/L and a control and a solvent control. The mean measured concentration (calculated as the average over all measurements) was 0.09 µg/L (45% of nominal) for the highest concentration. Taking into account the survival rates and the reproduction rates of the test animals, the 21-day NOEC was determined to be nominal 0.20 µg/L (mean measured 0.09 (µg/L).