Ethyl enantate

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:

2016-10-12 to 2016-10-15

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: all concentrations and control, after 0, 24, 48 and 72h
- Sample storage: stored at ambient temperature.
- Sample preparation: dilution with methanol. Last dilution in SPME vial containing 100 g/L NaCl and 50 µL internal standard.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- stock solution: 50 mg/L, stirred for 24h with 1100 rpm at room temperature, pH value adjusted to 8.1 with 10 M NaOH
- geometrical series: spacing factor 3.16: 0.158, 0.500, 1.58, 5.00, 15.8, 50 mg/L

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: HINDAK, SAG 61.81
- Source: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen
- Age of inoculum (at test initiation): 4 day old preculture
- Method of cultivation: fresh stocks prepared monthly, 2567 to 5130 lux, for 24h per day, nutrient medium Z

Test type:

static

Water media type:

freshwater

Remarks:

OECD medium

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol Ca+Mg/L

Test temperature:

mean temperature: 21.5°C, min.: 21°C, max.: 22°C

pH:

pH at start: 8
pH at end (72h): between 7.6 (highest test concentration) and 9.44 (control)

Nominal and measured concentrations:

Nominal concentrations: 50, 15.8, 5, 1.58, 0.5, 0.158 mg/L
Measured concentrations: concentrations were assessed every 24h. At the end of the experiment measured concentrations were < LOQ (details on measured concentrations are presented under 'Any other informatoin on materials and methods'. The time-weighed average (TWA) concentrations were 23.2, 0.506, 0.138, 0.157, 0.101, 0.0534 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile headspace flasks, 59 mL, with aluminium tops with PTFE seals
- Type: closed
- Headspace: no headspace
- Initial cells density: nominal approx. 5000 - 50000 cells/mL. Measured: 6305 cells/mL
- Control end cells density: 731589 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates):6

GROWTH MEDIUM
- Standard medium used: yes, prepared according to guideline


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline

OTHER TEST CONDITIONS
- Adjustment of pH: pH set at 8.1 at start of experiment, no further correction afterwards.
- Photoperiod: 24h/d light
- Light intensity and quality: 4440 to 8880 lux
- Agitation: rotary shaker at 70 rpm


EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: fluorimeter, excitation at 436 nm and emission at 685 nm;
- Observations: microscopic evaluation at start and end of the exposure


TEST CONCENTRATIONS
- Spacing factor for test concentrations of the final test: √10 (3.16)

Range finding study
- Test concentrations: 100, 10, 1 and 0.1 mg/L
- Results used to determine the conditions for the definitive study:
Nominal concentration (mg/L) % growth rate inhibition % yield inhibition
100 100 100
10 35 80
1 14 18
0.1 5 19

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.101 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.138 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.182 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 0.167-0.200

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.251 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 0.233-0.271

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.44 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 0.425-0.455

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.101 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.138 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.103 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Remarks on result:

other: 95% confidence interval: 0.0808-0.118

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.133 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Remarks on result:

other: 95% confidence interval: 0.122-0.145

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.202 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Remarks on result:

other: 95% confidence interval: 0.182-0.248

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no morphological abnormalities observed.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50 (growth rate): 0.435 mg/L with head space, 0.811 mg/L without headspace

Reported statistics and error estimates:

Sigmoidal dose-response curve was fitted to the data. R2 = 0.9985, P-value: 0.1356.
Normal distribution of the data (Shapiro-Wilk test and Levene's test)
The NOEC value was verified using the multiple sequential-rejective Welsh-t-test after Bonferroni-Holm

The exposure concentrations were assessed every 24h. As there is a decrease in test solution concentration with time, time-weighed average concentrations were used.

nominal concentration	Measured concentration (mg/L)
(mg/L)	0h	24h	48h	72h	TWA (mg/L)
50	43	42.8	47.3	0.881	23.2
15.8	12.7	14.1	0.0364	< LOQ	0.506
5	4.4	3.53	< LOQ	< LOQ	0.138
1.58	1.35	1.29	0.036	< LOQ	0.157
0.5	0.497	0.432	0.048	< LOQ	0.101
0.158	0.157	0.135	0.0402	< LOQ	0.0534
control	< LOQ	< LOQ	< LOQ	< LOQ

The cell densities were assessed and growth rate and yield calculated. The table below gives an overview of the means obtained:

TWA concentration (mg/L)	growth rate (d-1)	% inhibition	Yield (cells/mL)	% inhibition
23.2	n.a.	100	n.a.	100
0.506	0.657	59	39251	95
0.138	1.48	6	533259	26
0.157	1.48	7	522810	28
0.101	1.57	1	689629	5
0.0534	1.57	1	695145	4
Control	1.58		725284

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test substance towards freshwater green algae was assessed according to OECD 201. The test substance was found to inhibit the algae after 72h exposure. The EC50, EC10 and NOEC value derived based on growth rate were 0.440 mg/L, 0.182 mg/L and 0.101 mg/L respectively, based on time-weighed average concentrations.

Executive summary:

The toxicity of the test substance towards freshwater green algae, Pseudokirchneriella subcapitata was determined according to OECD 201. The green alga were exposed for 72h in static test set-up. Due to the volatility of the test item, glass flasks without headspace were used.

The alga were exposed to nominal concentrations: 0.158, 0.5, 1.58, 5, 15.8 and 50 mg/L. With time the concentration of the test substance decreased significantly, hence, time-weighed averages were used for reporting the results. The TWA concentrations were: 0.0534, 0.101, 0.157, 0.138, 0.506 and 23.2 mg/L.

The test substance was found to inhibit growth of alga. Based on growth rate, the EC50 was found to be 0.44 mg/L (0.425 -0.455), EC10 0.182 mg/L and NOEC 0.101 mg/L. When expressed as yield inhibition, the EC50 is 0.202 mg/L, EC10 0.103 mg/L and NOEC 0.101 mg/L.

Description of key information

The toxicity of the test substance towards freshwater green algae was assessed in one study. The study was performed according to OECD 201. The test substance was found to inhibit the algae after 72h exposure. The EC50 and EC10 value derived based on growth rate were 0.440 mg/L and 0.182 mg/L, respectively, time-weighed average concentration.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.44 mg/L

EC10 or NOEC for freshwater algae:

0.182 mg/L

Additional information

There is one study available in which the toxicity of the test substance towards freshwater green algae,Pseudokirchneriella subcapitata was determined according to OECD 201.The green alga were exposed for 72h in static test set-up. Due to the volatility of the test item, glass flasks without headspace were used.

The alga were exposed to nominal concentrations: 0.158, 0.5, 1.58, 5, 15.8 and 50 mg/L. With time the concentration of the test substance decreased significantly, hence, time-weighed averages were used for reporting the results. The TWA concentrations were: 0.0534, 0.101, 0.157, 0.138, 0.506 and 23.2 mg/L.

The test substance was found to inhibit growth of alga. Based on growth rate, the EC50 was found to be 0.44 mg/L (0.425 -0.455), EC10 is 0.182 mg/L and NOEC 0.101 mg/L. When expressed as yield inhibition, the EC50 is 0.202 mg/L, EC10 is 0.103 mg/L and NOEC 0.101 mg/L.