Reaction mass of 4-tert-butyl-2,5-xylenol and 6-tert-butyl-2,4-xylenol

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 8 to 19, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Meets the requirements of GLP. There are no deviations from the recommended guideline that could affect the outcome of the study. One minor deviation is stated.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

(the temperature lay in range of 21-25 ºC and therefore, higher than stated in the guideline. Because the controls showed normal exponential growth, this can be stated as uncritical)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

(the temperature lay in range of 21-25 ºC and therefore, higher than stated in the guideline. Because the controls showed normal exponential growth, this can be stated as uncritical)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Acetone was used as solvent to prepare the test solutions of the amount needed. A stock solution containing 100 g/L in acetone was prepared. From this stock solution, additional solutions (2.2/4.6/10/22/46 g/L) were prepared through dilution with acetone.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): the concentration of solvent in the test solutions was 0.1 mL/L at the most.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: unicellular freshwater green alga.
- Strain: CHODAT
- Source (laboratory, culture collection): MBM Sciencebridge GmbH (Institut für Pflanzenphysiologie of Universität Göttingen).
- Method of cultivation: the algae are kept as stock culture on solid agar at 8 °C. From an aliquot of this stock culture the pre-culture was prepared.

ACCLIMATION
- Culturing media and conditions (same as test or not): the nutrient-medium is 10-fold concentrated the pre-culture-medium
- Any deformed or abnormal cells observed: before usage, the culture was checked on the absence of cell aggregates

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 – 25 °C

pH:

7.3-7.9

Nominal and measured concentrations:

Table 9.1 d Measured Concentrations: please, see Any other information on materials and methods.
The measured concentrations ranged from 56 to 88 % of the nominal concentration at the beginning and from 27 to 52 % of the nominal concentration after 72 hours. The recovery after 72 hours was in a range of 38 to 84 % of the start concentration.
Therefore the determination of the biological results was based on the geometric means of the measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks, nominal volume 50 mL
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: no headspace

Main study:
- No. of vessels per concentration (replicates): 3 replicates for each treatment
- No. of vessels per control (replicates): 6 replicates
- No. of vessels per solvent control (replicates): 6 replicates
Reference study:
- No. of vessels per control (replicates): 6 replicates for the control
- No. of vessels per positive control (replicates): 3 replicates for each treatment

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: pH was adjusted to 7.0 (± 0.2) with HCl
- Light intensity and quality: 4440 – 8880 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: the absorption values of each treatment and control was determined by photometric measurement

TEST CONCENTRATIONS
- Test concentrations: 0.22 / 0.46 / 1.0 / 2.2 / 4.6 / 10 mg/L in acetone

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval not determinable

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval not determinable

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence interval not determinable

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval not determinable

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval not determinable

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence interval not determinable

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 5.5-36 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence interval: 1.7-3.8 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence interval: 1.7-4.3 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): in the microscopical observation in the main study , at a nominal concentrations of 2.2, 4.6 and 10 mg/L, smaller cells were found.
- Other: to avoid sedimentation of the algal cells, a marble was put into the test vessels

Results with reference substance (positive control):

- Results with reference substance valid? yes
Biological Results of the Positive Control:
Parameter Value 95%-confidence-interval
NOEC 72 h 0.16 mg/L not determinable
LOEC 72 h 0.25 mg/L not determinable
72h ErC50 1.8 mg/L 1.4 – 2.3 mg/L
72h EbC50 0.55 mg/L 0.48 – 0.62 mg/L
72h EC50 0.35 mg/L 0.30 – 0.40 mg/L

Reported statistics and error estimates:

As the test item is volatile, the test was performed in closed glass vessels without headspace. The measured concentrations at the end of the test were lower than at the beginning. This might be caused by adsorption of test item on algae cells and / or loss of test item during sampling for analytic determination and photometric measurement. Therefore the determination of the biological results was based on the geometric means of the measured concentrations.
For the estimation of the EC50s of the test item, the fits showed good statistical correspondence of the data with the dose-response-equation for the treatments which showed significant inhibition of algal growth. All three endpoints could be determined with acceptable confidence intervals.
The EC50s of potassium dichromate were determined in a current reference test. For the estimation of the EC50s of the positive control, the fits showed good statistical correspondence of the data with the dose-response-equation. The values were within the normal range of the laboratory.
The result of the test can be considered valid.

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were met.

Conclusions:

The following results for the test item were determined:

Endpoint NOEC LOEC EC50
Growth Rate 1.3 mg/L 3.0 mg/L 8.7 mg/L
AUC 1.3 mg/L 3.0 mg/L 2.7 mg/L
Yield 1.3 mg/L 3.0 mg/L 2.8 mg/L

[1]AUC1 (Area Under Curve according to EU Method C.3) means the integral of the biomass.
[2]Yield (according to OECD Guideline 201) is defined as the biomass at the end of the exposure period minus the biomass at the start of the exposure period.

Executive summary:

Two experiments were performed. In the first experiment, open glass flasks were used as test vessels and the content of test item was measured in filtered solutions. The result of the experiment showed that the test item is volatile and adsorption effects from test item on the membrane filters were possible. Therefore, the study was repeated in closed test vessels without headspace and analytical determination was performed with unfiltered solutions. The result of the first experiment isn’t reported; the data is kept with the raw data.

 

The study was performed using six concentrations ranging from 0.22 to 10 mg/L (nominal). The treatments were used to incubate the unicellular freshwater green alga Desmodesmus subspicatus for a period of 72 hours. The cell concentration of each replicate was determined by measuring the absorption of the cuvettes at 440 nm every 24 hours with a spectral photometer. With these measured values, the number of cells was calculated (linear correlation between cell concentration and absorption given). Then the growth rate µ, the area under the growth curve (AUC) and the Yield were determined. At the start and at the end of the test, the content of the test item in the test solutions was determined using GC. The recovery after 72 hours was in a range of 38 and 84 % of the start concentration, the measured concentration at the beginning of the test between 56 % and 88 % and at the end of the test between 27 % and 52 % of the nominal concentration, respectively. The lower recovery at the end of the test might be caused by adsorption of test item on to the algal cells and loss of test tem during sample preparation. Therefore, the determination of the results was based on the mean of the measured concentrations. The EC50s of potassium dichromate were tested in a current reference test. The values lay within the normal range of the laboratory. The following results for the test item were determined:

 

Endpoint	NOEC	LOEC	EC50
Growth Rate	1.3 mg/L	3.0 mg/L	8.7 mg/L
AUC	1.3 mg/L	3.0 mg/L	2.7 mg/L
Yield	1.3 mg/L	3.0 mg/L	2.8 mg/L

 

Description of key information

Key study: Experimental result: Test according to OECD guideline 201 and EU method C.3. GLP study.

The following results for the test item were determined:

Endpoint        NOEC            LOEC          EC50

Growth Rate   1.3 mg/L     3.0 mg/L    8.7 mg/L

AUC                 1.3 mg/L     3.0 mg/L    2.7 mg/L

Yield                1.3 mg/L      3.0 mg/L    2.8 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

8.7 mg/L

EC10 or NOEC for freshwater algae:

1.3 mg/L

Additional information

Key study: Experimental result: Test according to OECD guideline 201 and EU method C.3.

The following results for the test item were determined:

Endpoint NOEC LOEC EC50

Growth Rate 1.3 mg/L 3.0 mg/L 8.7 mg/L

AUC 1.3 mg/L 3.0 mg/L 2.7 mg/L

Yield 1.3 mg/L 3.0 mg/L 2.8 mg/L