Isopentyl salicylate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentration levels, the solvent control and the control
- Sampling frequency: after 0 (start of exposure), 24, 48 and 72 hours (end of the exposure). Separate replicates for each test item concentration and control for the test item analysis at the beginning of the exposure were prepared with algae.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solutions were prepared in measuring flasks (10 mL capacity). The test item was pipetted into the measuring flasks and filled up with the solvent. Therefore, the density was taken into account. The stock solutions were agitated. The test concentrations were prepared by adding 0.1 mL/L of the appropriate stock solutions to the dilution water. These solutions will be agitated and used for testing.
- Controls: Control and solvent control
- Chemical name of vehicle: Acetone
- Concentration of vehicle in test medium: Acetone was used to achieve the maximum solubility. Preliminary test with the slow stirring method were carried out. This method was not suitable to prepare concentrations near the maximum water solubility. Stirring phases of several days would be necessary and could also produce relevant concentrations of the degradation products. 0.1 mL/L in the stock solution, final test solutions and solvent control
- Evidence of undissolved material: none

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: HINDAK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG) Pflanzenphysiologisches Institut der Universität Göttingen; 37073 Göttingen, Germany: Cultivated at test facility.
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux for 24 hours per day.
- Culture medium: Nutrient medium Z according to Lüttge et al. (1994)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.5 °C

pH:

7.6 - 8.7

Nominal and measured concentrations:

Nominal test concentrations: control - 0.0200 - 0.0632 - 0.200 - 0.632 - 2.00 - 6.32 mg/L
TWA concentrations: < LOQ - 0.0172 - 0.0331 - 0.0960 - 0.247 - 0.742 - 2.71 mg/L

Details on test conditions:

TEST SYSTEM
- Type: open
- Material, size, fill volume: Sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton
wool plugs, 100 mL test volume
- Initial cells density: 6839 cells/ml
- Control end cells density:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Threefold concentrated AAP medium according to the guidelines
- Culture medium different from test medium: no
- Intervals of water quality measurement:
- pH: at the start of exposure were measured in one additional replicate of each test item concentration, solvent control and the control. At the end of exposure, the pH-values were
measured from pooled samples of each test item concentration, solvent control and the control
- Room temperature: was measured continuously
- Light intensity: prior to the start of exposure

OTHER TEST CONDITIONS
- Photoperiod: 24 hours/day light
- Light intensity and quality: Mean light intensity: 5656 lux (range: 5005 to 6053 lux)


EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm.
- Microscopic evaluation: Microscopic evaluation of the cells at the start and the end of the incubation period was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: yes (
- Test concentrations: 100, 10, 1 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.12 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.9 47-1.6 1 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.442 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.228 - 0.602 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.247 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.298 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.041 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.017 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): yes, > 232-fold
- Any stimulation of growth found in any treatment:none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none

Results with reference substance (positive control):

ErC50 (72 h) = 0.559 mg/L
EyC50 (72 h) = 0.32 mg/L

Reported statistics and error estimates:

- NOEC and LOEC were determined by calculation of analysis statistically significant differences of growth rates and yield
- Statistical tests:
- Shapiro-Wilk’s test on normal distribution was carried out with a significance level 0.01.
- Monotonicity was done by trend analysis by contrasts (significance level 0.05) for growth rate.
- Levene’s test on variance homogeneity was carried out with a significance level 0.01.
- Multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm was calculated with a significance level of 0.05 for yield.
- William’s Multiple Sequential t-test on significant differences was done with a significance level 0.05 for growth rate.

The difference between control and solvent control was determined by calculation of statistically significant differences of growth rates
and yield. A t-test was used for calculations. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

Mean cell densities

Time weighted average test item concentration	Cell density [cells/mL]
[mg/L]	0 hours	24 hours	48 hours	72 hours
2.71	6839	n.a.	n.a.	n.a.
0.742	6839	14037	69213	533566
0.247	6839	18654	109348	878434
0.0928	6839	23506	160272	1453705
0.0331*	6839	19248	92786	577758
0.0172	6839	31123	239392	1730348
Solvent Control	6839	34605	252808	1784587
Control	6839	29683	199879	1586058

n.a. = not applicable (Limit of quantification of the cell density (2736 cells/mL).

* This concentration is not taken into account due to a contamination of the measuring cylinder that was used for the preparation of this treatment.

Calculated Concentrations of the Test Item from the Measured Concentration of the Metabolite Isoamyl alcohol and the Test item

Values presented as Isoamyl salicylate

Sampling date	2017-07-24 0 h			2017-07-25 24 h			2017-07-26 48 h
Start of analysis	2017-07-24			2017-07-25			2017-07-26
Nominal test item con-centration [mg/L]	Conc. [mg/L]			Conc. [mg/L]			Conc. [mg/L]
	1)	2)	3)	1)	2)	3)	1)	2)	3)
6.32	4.60	0.0156	4.62	3.41	0.103	3.52	2.17	0.148	2.32
2.00	1.59	0.00591	1.60	1.06	0.0317	1.09	0.527	0.0329	0.560
0.631	0.654	0.00591	0.660	0.331	0.0143	0.345	0.160	0.0172	0.177
0.200	0.202	0.00591	0.208	0.130	0.00932	0.140	0.0584	0.0148	0.0732
0.0632 *	0.0527	0.00591	0.0586	0.0347	0.0106	0.0453	0.0174	0.00885	0.0262
0.0200	0.0181	0.00591	0.0240	0.0108	0.00868	0.0195	0.00748	0.00954	0.0170

continued

Sampling date	2017-07-27 72 h
Start of analysis	2017-07-27			Time Weighted Average Test Item Concentration [mg/L]
Nominal test item concentration [mg/L]	Conc. [mg/L]
	1)	2)	3)
6.32	1.15	0.146	1.30	2.71
2.00	0.234	0.0467	0.280	0.742
0.631	0.0836	0.00830	0.0919	0.247
0.200	0.0301	0.00591	0.0360	0.0960
0.0632 *	0.00999	0.00591	0.0159	0.0331
0.0200	0.00400	0.00591	0.00991	0.0172

Conc.  = Concentration of the test item (enrichment and dilution factor taken into account)

1)       = Measured concentration of test item

2)        = Calculated concentration of test item from measured concentration of the metabolite Isoamyl alcohol calculated with molar ratio to test item; (molar ratio = M(test item) / M(Isoamyl alcohol)

3)       = Summarized concentration of test item

*         = This concentration is not taken into account due to a contamination of the measuring cylinder that was used for the preparation of this treatment.

Validity criteria fulfilled:

yes

Conclusions:

The EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 1.12 mg/L and 0.296 mg/L, respectively. The EC10-values for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 0.442 mg/L and 0.0407 mg/L, respectively.

Description of key information

ErC50 (72 h) = 1.12 mg/L (TWA;Pseudokirchneriella subcapitata; OECD 201)

ErC10 (72 h) = 0..442 mg/L (TWA; Pseudokirchneriella subcapitata; OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

1.12 ng/L

EC10 or NOEC for freshwater algae:

0.442 ng/L

Additional information

For this endpoint one test is available. The influence of test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a study conducted according to OECD Guideline 201 and GLP.

The study was performed under static conditions. For preparing the sock solutions a slow stirring procedure was not appropriate since the test item hydrolysis fastly. Therefore, to achieve the maximum solubility of the test item Acetone was used as a solvent. Six test item concentrations were tested in a geometrical series with a dilution factor of 3.16: 0.0200 - 0.0632 - 0.200 - 0.632 - 2.00 - 6.32 mg/L. All concentration levels and the (solvent) control were analytically verified via GC-MS after 0 (start of exposure), 24, 48 and 72 hours (end of the exposure). Analytical monitoring also comprised the degradation product Isoamyl alcohol.

Using the sum of the measured concentrations of the test item and the calculated amount resulted from the metabolite (molecular ratio was taken into account) the time weighted average (TWA) concentrations were calculated (0.0172 – 0.0331 – 0.0960 – 0.247 – 0.742 – 2.71 mg/L).

The EC50-values with for inhibition of growth rate and yield after 72 hours were 1.12 mg/L and 0.296 mg/L, respectively. The EC10-values for inhibition of growth rate and yield after 72 hours were 0.442 mg/L and 0.0407 mg/L, respectively.