Ethyl 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

Study period:

From 2017-06-06 to 2017-06-13

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)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Initial concentrations: 0.250 - 0.790 - 2.50 - 7.90 - 25.0 - 79.0 mg/L, and a control
- Sampling method: All concentration levels and the controls were analytically verified via HPLC-DAD at the start (0 hours) and at the end of the exposure (72 hours) with algae. The highest test item concentration was analysed at the start and the end of the exposure without algae.
- Sample storage conditions before analysis: All original samples were stored at at 6 ± 2 °C.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution with a nominal loading of 79 mg test item/L was prepared with dilution water and stirred for 15 minutes at room temperature, 1100 rpm.Test concentrations were acquired by further dilution of the stock solution.
- Test concentrations: 0.250 - 0.790 - 2.50 - 7.90 - 25.0 - 79.0 mg/L
- Control: Six replicates (without test item) were incubated under the same conditions as the test concentrations

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: KORSHIKOV, SAG 61.81
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 - 5130 lux for 24 hours per day

ACCLIMATION
- Acclimation period: four days
- Culturing media and conditions (same as test or not): Nutrient medium Z, same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

22.5 – 23.5 °C, with mean 23°C

pH:

7.53 to 7.59 at test start and 8.08 to 8.97 at test end

Nominal and measured concentrations:

Nominal concentrations: 0.250 - 0.790 - 2.50 - 7.90 - 25.0 - 79.0 mg/L
Measured initial concentration: 0.242 - 0.759 - 2.33 - 7.60 - 23.8 - 76.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks
- Material, size, headspace, fill volume: volume: 250 mL, sealed with cotton wool plugs, fill volume: 100 mL
- Initial cells density: 6839 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, As recommended in OECD TG 201

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 24 hours/day light
- Light intensity and quality: range of the measured values: 4873 to 5923 lux, mean value: 5582 Lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Chlorophyll a-fluorescence
- Microscopic evaluation: at the start and the end of the incubation period;
- Physico-chemical Properties: pH-values at the start of exposure were measured in one additional replicate of each test item concentration and the control. At the end of exposure, the pH-values were measured from pooled samples of each test item concentration and the control. The room temperature was measured continuously. Light intensity was measured prior to the start of exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study
- Test concentrations: 0.500, 5.00, 50.0 mg/L
- Results used to determine the conditions for the definitive study: The stock solution and the dilution levels of 0.500 and 5.00 mg/L were visually clear throughout the exposure period. Growth rate inhibitions were 4%, 4%, 100% for 0.500, 5.00, 50.0 mg/L, respectively.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.89 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 4.7 - 16.6 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.47 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 7.65-17.4 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.33 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.68 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yoield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.31 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Microscopic evaluation: revealed no morphological abnormalities.
- Physicochemical Data: The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits. The test item solutions were visually clear throughout the exposure phase.
- Chemical analysis of test item concentrations: The measured concentrations of the test item were between 93% and 101% at the start of the exposure (0 hours) and between < LOQ and 53% at the end of the exposure (72 hours).

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: most recent test: growth rate 0.435 mg/L (95% CI: 0.414-0.458), yield 0.230 mg/L (95% CI: 0.179-0.322)

Results non-GLP range finder study:

Nominal concentration (mg/l)	Growth rate inhibition (%)	Yield inhibition (%)
50	100	100
5	4	18
0.5	4	20

Mean cell densities:

Measured initital test concentration (mg/L)	0h	24h	48h	72h
76	6839	n.a.	n.a.	n.a.
23.8	6839	3729	n.a.	n.a.
7.6	6839	27805	285829	1885138
2.33	6839	42967	471677	2663220
0.759	6839	44515	482273	2720238
0.242	6839	48064	494414	2715478
Control	6839	48920	50915-9	2832596

Evaluation of growth rate and yield inhibition after 72 hours:

Measured initial test item concentration [mg/L]	Replicate No.	Growth rate [d^-1]	Inhibition of growth rate [%]	Yield [cells/mL]	Inhibition of yield [%]
76	1	n.a.	100	n.a.	100
	2	n.a.	100	n.a.	100
	3	n.a.	100	n.a.	100
	Mean	(+) n.a.	100	(+) n.a.	100
23.8	1	-0.295	100	-4020	100
	2	n.a.	100	n.a.	100
	3	n.a.	100	n.a.	100
	Mean	(+) n.a.	100	(+) n.a.	100
7.6	1	1.88	6	1922130	32
	2	1.87	7	1850443	35
	3	1.87	7	1862323	34
	Mean	(+) 1.87	7	(+) 1878299	34
2.33	1	2.00	1	2731755	3
	2	2.00	0	2755748	2
	3	1.97	2	2481640	12
	Mean	(+)* 1.99	1	(-) 2656381	6
0.759	1	2.00	0	2778512	2
	2	1.99	1	2628467	7
	3	2.00	1	2733218	3
	Mean	(-) 2.00	1	(-) 2713399	4
0.242	1	2.02	-1	2927094	-4
	2	2.01	0	2829132	0
	3	1.95	3	2369691	16
	Mean	(-) 1.99	1	(-) 2708639	4
Control	1	2.01		2867579
	2	2.03		3006213
	3	2.00		2784657
	4	2.01		2800047
	5	2.00		2718939
	6	2.00		2777108
	Mean	2.01		2825757

n.a. = not applicable

*= statistically significant, but biologically not relevant (inhibition < 5%)

TOC content of the test media at the start and the end of the exposure:

Measured initial test item concentration [mg/L]	TOC [mg C/L]
	Test start (without algae), 0 hours	Test end (without algae), 72 hours
76	49.4	34
23.8	20.5	12.8
Control	< 2.00	< 2.00

Limit of quantification = 2.00 mg C/L

Measured Concentrations and Percentage of the Nominal Concentration of test item in Fresh Medium (0hours) and Old Medium (72hours) with Algae:

Sample	0 hours fresh medium		72 hours old medium
Nominal concentration of the test item [mg/L]	Meas. conc. [mg/L]	[%]	Meas. conc.  [mg/L]	[%]
79.0^1)	80.1	101	42.1	53
79	76	96	36.7	46
25.00	23.8	95	9.13	37
7.90	7.6	96	< LOQ
2.50	2.33	93	0.119	5
0.79	0.759	96	< LOQ
0.25	0.242	97	< LOQ
Control	< LOQ		< LOQ

Meas. Conc. = Measured concentration of the test item (dilution factor taken into account), mean value oftwo injections

%= Percentage of the nominal concentration of the test item

LOQ= Limit of quantification (0.100 mg test item/L)

¹⁾= replicate without algae

Validity criteria fulfilled:

yes

Conclusions:

In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on measured initial test item concentrations): EC50-values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 9.47 mg/L and 8.68 mg/L, respectively. The EC10-values ErC10 and EyC10 were 7.89 mg/L and 5.31, respectively.

Executive summary:

The toxicity of test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and GLP to assess effect on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. The main test was carried out with six concentrations in a geometrical series with a dilution factor of 10: 0.250 - 0.790 - 2.50 - 7.90 - 25.0 - 79.0 mg/L.

The concentrations of the test item in all test concentrations and the control were analytically verified by HPLC- DAD at the start and the end of exposure. Additionally the two highest concentration levels and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start and at the end of the exposure. The measured concentrations of test item in the fresh media (0 h) were between 93 and 97% of the nominal values. The measured concentrations in the old media (72 h) were in the range of < LOQ to 53% of the nominal values. Therefore, the evaluation based on measured initial concentrations was considered the most fitting evaluation.

In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata fter 72 hours with the following effect values (based on measured initial test item concentrations): EC50 -values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 9.47 mg/L and 8.68 mg/L, respectively. The EC10 -values ErC10 and EyC10 were 7.89 mg/L and 5.31, respectively.

Description of key information

ErC50 (72 h) = 9.47 mg/L (measured, Pseudokirchneriella subcapitata, OECD 201)

ErC10 (72 h) = 7.89 mg/L (measured, Pseudokirchneriella subcapitata, OECD 201)

Key value for chemical safety assessment

EC50 for freshwater algae:

9.47 mg/L

EC10 or NOEC for freshwater algae:

7.89 mg/L

Additional information

The toxicity of test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and GLP to assess effect on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. The main test was carried out with six concentrations in a geometrical series with a dilution factor of 10: 0.250 - 0.790 - 2.50 - 7.90 - 25.0 - 79.0 mg/L.

The concentrations of the test item in all test concentrations and the control were analytically verified by HPLC- DAD at the start and the end of exposure. Additionally the two highest concentration levels and the control were analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start and at the end of the exposure. The measured concentrations of test item in the fresh media (0 h) were between 93 and 97% of the nominal values. The measured concentrations in the old media (72 h) were in the range of < LOQ to 53% of the nominal values. Therefore, the evaluation based on measured initial concentrations was considered the most fitting evaluation.

In this study, the test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata fter 72 hours with the following effect values (based on measured initial test item concentrations): EC50 -values for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 9.47 mg/L and 8.68 mg/L, respectively. The EC10 -values ErC10 and EyC10 were 7.89 mg/L and 5.31, respectively.