Reaction mass of isopentyl salicylate and 2-methylbutyl salicylate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 26th, 2018 to March 8th, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 18,25,35,50,70,100 mg/L
- Sampling method: The concentration of the test item in the control and the solutions was checked by HPLC/DAD at the start (t=0h) and at the end of the test (t=48h).
- Sample storage conditions before analysis: All the solutions were kept in the refrigerator in order to be analysed but not used.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preliminary works (non GLP) were performed to define the solution preparation protocol and to obtain the maximum achievable dissolved concentration of the test item in the test medium. A study of the stability of the test item was carried out under the testing conditions. According to the preliminary test, the OECD 202 was performed in semi-static conditions. Three independent stock solutions at 100 mg/L (nominal concentration) were stirred 24 hours at 20 ± 2ºC and at darkness. After this step, the samples were filtered on cellulose acetate (filter porosity: 0,45 μm) after a settling period of 60 minutes. Chemical analysis was performed and the stock solution with the highest measured concentration was chosen for the preparation of the range by serial dilution of this stock solution.
- Controls: concurrent untreated control (4 replicates) was examined in parallel to the test item concentrations. Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Daphnia magna Straus, clone number 5
- Source: Laboratoire de Chimie-Écotoxicologie, FCBA (Institut Technologique Forêt Cellulose Bois Construction Ameublement), Pôle des Laboratoires Bois, Allée de Boutaut – BP 227, 33028 BORDEAUX Cedex - FRANCE
- Feeding during test: none

ACCLIMATION
- Acclimation conditions (same as test or not): The daphnids breeding was carried out in closed bottles placed in a climatic chamber free from any toxic vapour and in medium M4.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Daphnids used in this experimentation were previously filtered in order to obtain animals from a size between 560 μm and 800 μm (corresponding to daphnids less than 24 hours old).

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

Dilution water M4: 270

Test temperature:

19.7-20 ºC

pH:

Dilution water M4: 8.2-8.4
Final test: 7.7-7.8

Dissolved oxygen:

Dilution water M4:8.7-9.7
Final test: 8.9

Nominal and measured concentrations:

Nominal concentrations: 18, 25, 35, 50, 70, 100 mg/l
Measured concentrations a T0: (Before Renewal) <0.5, 0.212, 0.282, 0.392, 0.527, 1.150, 1.46// (After Renewal) <0.5, 0.602, 0.815, 0.986, 1.351, 1.820, 3.222 mg/l
Measured concentrations a T48: <0.5, 0.290, 0.498, 05.90, 1.146, 1.848, 2.763

Details on test conditions:

TEST SYSTEM
- Test vessel: glass test tube
- Type: open
- Material, size, headspace, fill volume: 10 mL test tube
- 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: (M4 ELENDT) The medium was prepared from concentrated stock solutions in ultrapure water as described in the 202 OECD guideline.
- Intervals of water quality measurement: measured at T0

OTHER TEST CONDITIONS
- Adjustment of pH: The pH of the solutions remained in the range 6 – 9, the pH adjustment was not made with HCl and NaOH.
- Light intensity: Darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobilised daphnids, 24h, 48h

RANGE-FINDING STUDY
- Test concentrations: 1, 10, 100 mg/l
- Results used to determine the conditions for the definitive study: The result obtained from the test item in the preliminary test give an EC50 at 100 mg/L expressed in nominal concentrations.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 2.39 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

1.89 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

0.76 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: 0%
- Abnormal responses: Some signs of stress, reduced activity, were observed on the mobile daphnids after 24 and 48 hours of exposure to each nominal test item concentration.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ECx: EC50 (referce substance) at 24 hours is 1.40 mg/l

Reported statistics and error estimates:

Statistical significance was determined with the Dunnett test (α=5%) from raw data with the scientific software Minitab 17. A significant difference between the control was observed at the concentration of 14.1 mg/L.

Due to instability of the test item in test media (concentrations not maintained within ± 20% of the measured initial concentration), the immobility of the daphnids was determined in a closed semi-static 48-hours test by visual observation after 24 and 48 hours.

Table 1. Results summary

Observation time	24h	48h
EC10 in mg/l [Confidence Limit]	1.89 mg/L [0.94 - *]	0.76 mg/L [0.58 – 0.89]
EC50 in mg/l [Confidence Limit]	> 2.39 mg/L*	1.30 mg/L [1.13 – 1.53]

Table 2. Daphnids immobilisation.

Nominal test item concentration ( mg/L)	Number of daphnids exposed at the start	Immobilised daphnids (%)
		at 24h	at 48h
Control	20	0 (0%)	0 (0%)
10	20	1 (5%)	17 (85%)
18	20	4 (20%)	15 (75%)
32	20	4 (20%)	13 (65%)
56	20	5 (25%)	14 (70%)
100	20	3 (15%)	14 (70%)
180	20	6 (30%)	18 (90%)

Table 3. pH values during final test

Nominal test item concentration (% v/v)	pH
	T0 Fresh solution	T24h Old solution	T24h Fresh solution	T48 h Old solution
Control	7.9	7.6	8.0	7.7
18	8.0	7.6	8.1	7.7
25	8.0	7.7	8.1	7.7
35	8.0	7.6	8.1	7.7
50	8.0	7.7	8.1	7.7
70	8.0	7.7	8.1	7.7
100	8.0	7.7	8.1	7.8

Table 4. Dissolved oxygen values during the final test.

Nominal test item concentration (mg/L)	O2 (mg/L)
	T0 Fresh solution (Air: 9.7 mg/L)	T24h Old solution (Air: 9.7 mg/L)	T24h Fresh solution (Air: 9.7 mg/L)	T48 h Old solution (Air: 9.4 mg/L)
Control	8.8	8.6	8.8	8.7
18	8.6	8.5	8.6	8.7
25	8.6	8.5	8.6	8.6
35	8.7	8.4	8.6	8.5
50	8.6	8.6	8.5	8.6
70	8.6	8.6	8.6	8.6
100	8.5	8.6	8.5	8.6

Validity criteria fulfilled:

yes

Remarks:

There were 0% immobilised daphnids in the control and the dissolved oxygen (8.4 mg/l) was more than 3 mg/L

Conclusions:

The 48h-EC50 of the test item in Daphnia magna based on immobilisation was found to be 1.30 mg/L and the 24h-EC10 was >2.39 mg/L.

Executive summary:

An acute immobilisation test on Daphnia magna was performed in accordance with OECD Guideline 202, under GLP conditions. Based on the results obtained in a preliminary test, a final test at six nominal test item concentrations (18, 25, 35, 50, 70, 100 mg/L) and a control were performed. Test concentrations were analytically determined (validated method) at the beginning and at the end of the test. Due to the instability of the test item in test media (concentrations not maintained within ± 20 % of the measured initial concentration), the immobility of the daphnids was determined in a closed semi-static 48-hours test by visual observation after 24 and 48 hours. The test results were determined and expressed relative to the geometric mean of the measured concentrations. 20 animals were exposed to each concentration divided into 4 replicates with 5 animals in each 10 ml glass test tubes. The organisms were exposed to the test item or the control for 48 h and immobilisation was recorded at 24 and 48 h. Positive controls (potassium dichromate) are regularly performed to ensure the quality of the results. All validity criteria were met. The 48h-EC50 was determined to be 1.30 mg/L and the 24h-EC10 was >2.39 mg/L.

Description of key information

Key study. Method according to OECD 202, GLP study. The 48h-EC50 based on immobilisation was determined to be 1.30 mg/l.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.3 mg/L

Additional information

An acute immobilisation test on Daphnia magna was performed in accordance with OECD Guideline 202 , under GLP conditions. Based on the results obtained in a preliminary test, a final test at six nominal test item concentrations (18, 25, 35, 50, 70, 100 mg/L) and a control were performed. Due to instability of the test item in test media (concentrations not maintained within ± 20% of the measured initial concentration), the immobility of the daphnids was determined in a closed semi-static 48-hours test by visual observation after 24 and 48 hours. The test results were determined and expressed relative to the geometric mean of the measured concentrations. 20 animals were exposed to each concentration divided into 4 replicates with 5 animals in each 10 ml glass test tubes. The organisms were exposed to the test item or the control for 48 h and immobilisation was recorded at 24 and 48 hours. All validity criteria were met. The 48h-EC50 was determined to be 1.30 mg/l and the 24h-EC10 was >2.39 mg/L.