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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
13 April 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
p-tert-butylaniline
EC Number:
212-215-9
EC Name:
p-tert-butylaniline
Cas Number:
769-92-6
Molecular formula:
C10H15N
IUPAC Name:
p-tert-butylaniline

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.06, 0.14, 0.31, 0.68 and 1.50 mg/L (nominal)
- Sampling method: Samples of freshly prepared test media were taken from all five test solutions and the control at the beginning of the test. Samples of aged test concentrations were taken at the end of the test (48 hours) directly from pooled replicates per test concentration and control.
A volume of 7.5 mL of sample was used and all samples were stabilized with 7.5 mL of acetonitrile immediately after the sampling.

- Sample storage conditions before analysis: Samples were stored at -20°C in a refrigerator until shipment to the partner lab for analysis. For security reasons, a reserve set of stabilized samples was stored deep frozen.

Test solutions

Vehicle:
no
Remarks:
medium was used
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock and all test item solutions were prepared in Cu-reduced dilution water.
Before test start, an appropriate stock solution was prepared by weighing in 1.5 mg of test item and transferring it to 1L Cu-reduced dilution water. The stock solution also served as highest test concentration. The stock solution was stirred for about 24 hours at room temperature.
The other test concentrations were prepared by dilution of the stock solution with Cu-reduced dilution water using a spacing factor of 2.2.
- Controls: The control consisted of dilution water only.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none stated

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene. Specimens used in the test were bred in the laboratory of the Fraunhofer IME.
- Feeding during test: According to the OECD guideline 202, the daphnids were not fed during the test.

PRE-TREATMENT
Adult Daphnia, at least 3 weeks old, were separated from the stock population by sieving. Batches of 30 to 50 animals were held at room temperature in ca. 1.8 L dilution water for one week. During this week the daphnids were fed daily with an algal suspension (Desmodesmus subspicatus) and ArtemioFluid (JBL). Algae growing in the log-phase were centrifuged and the pellet was re-suspended in a few mL of medium. 30 mL of this suspension was given to 1 L Daphnia medium. The water was changed once per week. Newborn D. magna were separated by sieving, the first generation was discarded. Individuals applied in the test were transferred with a bore Pasteur pipette a few hours after sieving to ensure applying only healthy specimens.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
none

Test conditions

Test temperature:
18-22 °C (mean temperature of 20.5 °C)
pH:
8.1 – 8.5
Dissolved oxygen:
8.1 - 8.4 mg/L
Nominal and measured concentrations:
Nominal: 0.06, 0.14, 0.31, 0.68 and 1.50 mg/L
Measured: 0.08, 0.16, 0.33, 0.71 and 1.30 mg/L (fresh samples); 0.05, 0.11, 0.27, 0.50 and 1.44 mg/L (aged samples)
Geometric mean: 0.06, 0.13, 0.30, 0.60 and 1.37 mg/L (used for evaluation)
Details on test conditions:
TEST SYSTEM
- Test vessel: Numbered glass beakers (50 mL nominal volume) were used. The containers were filled up with about 50 mL test solution. The beakers were covered with glass panes to prevent from evaporation as much as possible, but also permit gaseous exchange between the medium and the atmosphere and access of light.
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): The main test was conducted as static test without renewal of test medium.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified drinking water was used as holding- and dilution water. The purification included filtration with activated charcoal, passage through a limestone column, and aeration. To avoid copper contamination, plastic water pipes are used for the testing facilities.
The following water chemistry data are recorded regularly in the testing facility and are reported: pH, conductivity, dissolved oxygen content, content of nitrate, nitrite, ammonium, phosphate, calcium, magnesium, total hardness, alkalinity, DOC content (or NPOC, as appropriate), content of metals (copper, iron, manganese and zinc).
In spite of the passage through a lime-stone column, hardness was below the recommended (not required) range (140 - 250 mg/L as CaCO3) for Daphnia magna indicated in the guideline. Historical data showed values of 70 – 120 mg/L as CaCO3. However, according to the guideline lower hardness may be also appropriate for other Daphnia species. Historical results from reference tests at the IME indicate that test conditions are also suitable for the Daphnia magna strain used at the IME.


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The objective of the study was the assessment of the effects of the test item on the mobility and other signs of intoxication in Daphnia magna in order to assess effects of the test substance to the aquatic environment. Young daphnids, aged less than 24 hours at the start of the test, were exposed to the test substance added to water at a range of five concentrations for 48 h under static conditions. At the end of the test, the total number of mobile specimens was assessed.

VEHICLE CONTROL PERFORMED: yes, dilution water only

RANGE-FINDING STUDY
- Test concentrations: A non-GLP range finder test was performed with the test item in order to determine the concentration range which should be applied in the definitive test. In the range finder test three nominal test concentrations of 1.0, 10.0 and 100 mg active substance (a.s.)/L were applied. Preparation of the test solutions was performed as for the main test for each of the test concentration using a spacing factor of 10. The control consisted of Cu-reduced dilution water only. The range-finder test was conducted with four replicates per concentration with five daphnids per beaker.
The test was performed under static conditions for 48 hours.
After 48 hours in the two highest test concentrations of 100.0 and 10.0 mg/L all daphnids died, resulting in 100 % mortality. In the lowest test concentration of 1.0 mg/L 75% of the daphnids were found to be dead. In the control no mortality was observed. The observed effects were statistically significant.
Reference substance (positive control):
yes
Remarks:
According to the guideline no reference item is required. However, the sensitivity of the test clone was checked twice a year in non-GLP tests by using K2Cr2O7 as reference substance.

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
LC10
Effect conc.:
0.52 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
0.98 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
1.37 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The study was performed according to OECD TG 202 without deviations, the results were so obtained via a scientifically reasonable method. Hence, there is no doubt that the obtained results are not reliable:
The test item p-tert. Butylanilin is acutely toxic to Daphnia magna under the chosen test conditions. The LC50 was calculated to be 0.98 mg/L The NOEC was calculated to be 0.60 mg/L and the LOEC was determined to be 1.37 mg/L.
Based on these results, the test item has to be classified as aquatic acute Cat. 1, M factor 1. Further, with regard to chronic toxicity and taking into account the facts that the test item is soluble in water and not readily biodegradable, the test item should be classified as aquatic chronic Cat. 1, M factor 1.
Executive summary:

A GLP study was performed to determine the influence of the test item p-tert. Butylanilin on immobilization of daphnids of the species Daphnia magna. For this, the daphnids were placed in water containing the test item in nominal loadings of 0.06, 0.14, 0.31, 0.68 and 1.50 mg per liter. The test was conducted under static conditions for 48 h. Effects on immobilization were determined after 24 and 48 hours according to OECD guideline 202. Samples for chemical analysis were taken of fresh and aged test solutions.

The nominal test concentrations were prepared in Cu-reduced dilution water. A stock solution of 1.50 mg/L was prepared and serially diluted to obtain the lower test concentrations.

 The concentrations of the test item in the media were confirmed by measurements of the test item concentrations before and after the test by HPLC-UV/VIS (LOQ = 0.031 mg/L). At test start measured concentrations of fresh media were 0.08, 0.16, 0.33, 0.71 and 1.30 mg/L, corresponding to 86.8 – 127% of nominal concentrations. At test end measured concentrations of aged samples were 0.05, 0.11, 0.27, 0.50 and 1.44 mg/L, corresponding to 72.9 – 96.0 % of nominal concentrations.

Since measured concentrations of fresh and aged test media were not in a range of 80 – 120 % of nominal concentrations, the geometric mean measured concentrations were used for the evaluation. Geometric mean concentrations of fresh and aged test media were 0.06, 0.13, 0.30, 0.60 and 1.37 mg/L.

 At the end of the test, a significantly increased immobility of daphnids was observed in the highest concentration of 1.37 mg/L when compared to the control daphnids. The calculated LC50 was determined to be 0.98mg/L. A NOEC of 0.60 mg/L and a LOEC of 1.37 mg/L were calculated. The test item p-tert. Butylanilin is acutely toxic to Daphnia magna under the chosen test conditions.