Isopropyl acetoacetate

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:

2020-06-11 to 2020-08-21

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:

Adopted March 23, 2006, corrected July 28, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

"Freshwater Algae and Cyanobacteria, Growth Inhibition Test ", Official Journal of the European Union (EN), dated August 24, 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The samples were taken from the biological phase of the study. Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment. All samples remained untreated. All samples were stored in a freezer (< - 20 °C), protected from light until analysis was performed.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test medium of the highest test concentration of nominal 100 mg test item/L was prepared by dissolving 96.8 mg test item into 968 mL test water by intense stirring for 25 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations. The test media were prepared just before introduction of the algae (= start of the test).

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Strain No. 61.81 SAG
- Source: Collection of Algal Cultures, 37073 Göttingen, Germany
- Cell density: 5000 algal cells /mL

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22.0 - 22.7 °C

pH:

8.2 - 8.9 (in the control group);
7.9 - 9.1 (in the treatment group)

Nominal and measured concentrations:

Nominal concentration: 100, 32, 10, 3.2 and 1.0 mg test item/L;
Measured concentration: mean of 93 % of nominal.

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 50 mL volume
- Type: closed
- Initial cells density: 5000 algal cells /mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity: 5222 lux (range: 4510 to 5940 lux)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: spectrophotometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16

RANGE FINDING STUDY
- Test concentrations: 100, 32, 10, 3.2 and 1.0 mg test item/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 93.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 93.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 93.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % conf. interval: 14.8 - 59.0 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

30.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

30.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

93.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

93.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of 100 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested

Results with reference substance (positive control):

- Results with reference substance valid? Yes

For the evaluation of the reliability of the test system and the experimental conditions Potassium dichromate is tested at least twice a year. The last study with the reference item Potassium dichromate was performed in January 2020 and the following results were obtained:
The 72h ErC50: 0.858 mg/L, (95% confidence limits: 0.811 - 0.919 mg/L)
The 72h EyC50: 0.470 mg/L, (95% confidence limits: 0.450 - 0.488 mg/L)

Biological Results

Table 1: Influence of the test item on the Growth of Pseudokirchneriella subcapitata

Parameter	Yield [mg test item/L]	Growth rate [mg test item/L]
72-hour EC50	> 93.9	> 93.9
95 % conf. interval	n.d.	n.d.
72-hour EC20	> 93.9	> 93.9
95 % conf. interval	n.d.	n.d.
72-hour EC10	27.0	> 93.9
95 % conf. interval	14.8 - 59.0	n.d.
72-hour NOEC	30.3	30.3
72-hour LOEC	93.9	93.9

 

n.d. = not determinable

Values refer to geometric mean measured test concentrations

 

Analytical Results

Table 2: Summary of Analytical Results

Sample Description [mg test item/L]	fresh (0h)			aged (24h)			aged (48h)			aged (72h)			Geometric Mean ^2,3
	% of nominal^1	RSD (%)	n	% of nominal^1	RSD (%)	n	% of nominal^1	RSD (%)	n	% of nominal^1	RSD (%)	n	(%)	(mg test item/L)
Control	< LOD	n.a.	2	< LOD	n.a.	2	< LOD	n.a.	2	< LOD	n.a.	2	n.a.	n.a.
1.0	94	5	2	93	4	2	87	8	2	65	9	2	86	0.859
3.2	100	0	2	98	2	2	92	14	2	80	9	2	93	2.98
10	108	2	2	105	1	2	92	1	2	92	1	2	99	9.86
32	98	2	2	99	2	2	93	2	2	88	0	2	95	30.3
100	98	0	2	96	2	2	93	4	2	89	1	2	94	93.9

 

¹ Mean value of all measured samples per treatment group

² Calculated according to OECD Guidance Document No. 23, Annex 2

³ The tabulated results represent results rounded to three significant digits

LOD: Limit of Detection (= 0.2 mg test item/L)

RSD: relative standard deviation per treatment group

n: number of analysed samples

n.a.: not applicable

 

Table 3: Algal Cell Densities during the Test Period of 72 Hours

 

Geometric mean measured concentration [mg test item/L]	Flask No.	Yield y [10000 cells/mL] after
		24 hours	48 hours	72 hours
Control	1	1.491	15.366	71.548
	2	1.946	21.735	113.628
	3	2.173	20.597	112.264
	4	2.173	19.233	120.680
	5	2.628	19.233	118.860
	6	2.173	19.233	116.130
	m	2.097	19.233	108.852
	s	0.371	2.148	18.542
0.859	1	2.173	20.597	116.585
	2	2.856	24.464	142.061
	3	2.401	21.507	131.370
	m	2.477	22.190	130.005
	s	0.347	2.022	12.792
2.98	1	1.491	16.276	100.436
	2	1.718	22.190	130.460
	3	1.946	18.323	119.997
	m	1.718	18.929	116.964
	s	0.227	3.003	15.240
9.86	1	2.628	23.100	138.194
	2	2.628	22.417	129.323
	3	2.401	21.735	131.598
	m	2.552	22.417	133.038
	s	0.131	0.682	4.608
30.3	1	2.628	20.143	130.688
	2	2.173	20.825	105.212
	3	2.401	17.868	99.753
	m	2.401	19.612	111.884
	s	0.227	1.548	16.511
93.9	1	2.401	19.460	86.333
	2	1.946	16.048	84.514
	3	1.946	15.593	85.878
	m	2.097	17.034	85.575
	s	0.263	2.113	0.947

m: mean value;

s: standard deviation At test start nominal 5000 algal cells/mL were inoculated

 

Table 4: Yield during the Test Period of 72 Hours

 

Geometric mean measured concentration [mg test item/L]	Flask No.	Yield y [10000 cells/mL] after
		24 hours	48 hours	72 hours
Control	1	0.991	14.866	71.048
	2	1.446	21.235	113.128
	3	1.673	20.097	111.764
	4	1.673	18.733	120.180
	5	2.128	18.733	118.360
	6	1.673	18.733	115.630
	m	1.597	18.733	108.352
	s	0.371	2.148	18.542
0.859	1	1.673	20.097	116.085
	2	2.356	23.964	141.561
	3	1.901	21.007	130.870
	m	1.977	21.690	129.505
	s	0.347	2.022	12.792
2.98	1	0.991	15.776	99.936
	2	1.218	21.690	129.960
	3	1.446	17.823	119.497
	m	1.218	18.429	116.464
	s	0.227	3.003	15.240
9.86	1	2.128	22.600	137.694
	2	2.128	21.917	128.823
	3	1.901	21.235	131.098
	m	2.052	21.917	132.538
	s	0.131	0.682	4.608
30.3	1	2.128	19.643	130.188
	2	1.673	20.325	104.712
	3	1.901	17.368	99.253
	m	1.901	19.112	111.384
	s	0.227	1.548	16.511
93.9	1	1.901	18.960	85.833
	2	1.446	15.548	84.014
	3	1.446	15.093	85.378
	m	1.597	16.534	85.075
	s	0.263	2.113	0.947

m: mean value; s: standard deviation

 

Table 5: Growth rate during the Test Period of 72 Hours

 

Geometric mean measured concentration [mg test item/L]	Flask No.	Yield y [10000 cells/mL] after
		24 hours	48 hours	72 hours
Control	1	1.491	15.366	71.548
	2	1.946	21.735	113.628
	3	2.173	20.597	112.264
	4	2.173	19.233	120.680
	5	2.628	19.233	118.860
	6	2.173	19.233	116.130
	m	2.097	19.233	108.852
	s	0.371	2.148	18.542
0.859	1	2.173	20.597	116.585
	2	2.856	24.464	142.061
	3	2.401	21.507	131.370
	m	2.477	22.190	130.005
	s	0.347	2.022	12.792
2.98	1	1.491	16.276	100.436
	2	1.718	22.190	130.460
	3	1.946	18.323	119.997
	m	1.718	18.929	116.964
	s	0.227	3.003	15.240
9.86	1	2.628	23.100	138.194
	2	2.628	22.417	129.323
	3	2.401	21.735	131.598
	m	2.552	22.417	133.038
	s	0.131	0.682	4.608
30.3	1	2.628	20.143	130.688
	2	2.173	20.825	105.212
	3	2.401	17.868	99.753
	m	2.401	19.612	111.884
	s	0.227	1.548	16.511
93.9	1	2.401	19.460	86.333
	2	1.946	16.048	84.514
	3	1.946	15.593	85.878
	m	2.097	17.034	85.575
	s	0.263	2.113	0.947

m: mean value; s: standard deviation

 

Table 6: Results of the Most Recent Test with Pseudokirchneriella subcapitata performed with the Reference Item Potassium Dichromate in January 2020

 

Parameter (0-72 h)	Yield [mg test item/L]	Growth rate [mg test item/L]	Biomass [mg test item/L]
72-hour EC50	0.470	0.858	0.495
95 % conf. interval	0.450 - 0.488	0.811 - 0.919	0.471 - 0.518
72-hour EC20	0.316	0.573	0.308
95 % conf. interval	0.289 - 0.339	0.540 - 0.603	0.277 - 0.334
72-hour EC10	0.256	0.464	0.240
95 % conf. interval	0.228 - 0.281	0.424 - 0.498	0.209 - 0.267
72-hour NOEC	0.20	0.20	0.063
72-hour LOEC	0.63	0.63	0.20

 

 

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on algal growth was assessed using the freshwater green alga Pseudokirchneriella subcapitata according to OECD TG 201 showed the following results: The 72-hour EyC50 was calculated to be > 93.9 mg test item/L and the 72-hour ErC50 value was calculated to be > 93.9 mg test item/L. The 72-hour EyC10 was calculated to be 27.0 mg test item/L (95% CI: 14.8 - 59.0 mg/L) and the 72-hour ErC10 value was calculated to be > 93.9 mg test item/L. The 72-hour NOEyC was determined to be 30.3 mg test item/L and the associated 72-hour LOEyC was 93.9 mg test item/L. The 72-hour NOErC was determined to be 30.3 mg test item/L and the associated 72-hour LOErC was 93.9 mg test item/L.

Executive summary:

The acute toxicity of the test item was investigated in a GLP-study according to OECD TG 201 using unicellular freshwater green alga Pseudokirchneriella subcapitata. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations (100, 32, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16)) of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. The test method and the test species Pseudokirchneriella subcapitata are recommended by the guideline. This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test media were inoculated with nominal 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometric measurement. The quantification of the test item in the test samples was performed using high performance liquid chromatography (HPLC) with UV detection. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. At the start of the test 100 % of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 83 % of the nominal value was determined (average of all test concentrations). Intermediate samples at 24-hours and 48-hours were taken to track the concentration decrease during the course of the test. During the test the algae were exposed to a mean of 93 % of the nominal concentration. All reported results refer to nominal values. Thus, the 72-hour E_yC₅₀ was calculated to be > 93.9 mg test item/L and the 72-hour E_rC₅₀ value was calculated to be > 93.9 mg test item/L. The 72-hour NOE_yC was determined to be 30.3 mg test item/L and the associated 72-hour LOE_yC was 93.9 mg test item/L. The 72-hour NOE_rC was determined to be 30.3 mg test item/L and the associated 72-hour LOE_rC was 93.9 mg test item/L. No abnormal appearance of the algal cells was observed during the experiment.

Description of key information

The effect of the test item on algal growth was assessed using the freshwater green alga Pseudokirchneriella subcapitata according to OECD TG 201 and showed the following results: The 72-hour E_yC₅₀ was calculated to be > 93.9 mg test item/L and the 72-hour E_rC₅₀ value was calculated to be > 93.9 mg test item/L. The 72-hour E_yC₁₀ was calculated to be 27.0 mg test item/L (95% CI: 14.8 - 59.0 mg/L) and the 72-hour E_rC₁₀ value was calculated to be > 93.9 mg test item/L. The 72-hour NOE_yC was determined to be 30.3 mg test item/L and the associated 72-hour LOE_yC was 93.9 mg test item/L. The 72-hour NOE_rC was determined to be 30.3 mg test item/L and the associated 72-hour LOE_rC was 93.9 mg test item/L (reference 6.1.5-1).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

30.3 mg/L

Additional information

The acute toxicity of the test item was investigated in a GLP-study according to OECD TG 201 using unicellular freshwater green alga Pseudokirchneriella subcapitata. For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations (100, 32, 10, 3.2 and 1.0 mg test item/L (spacing factor 3.16)) of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations. The test method and the test species Pseudokirchneriella subcapitata are recommended by the guideline. This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test media were inoculated with nominal 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 hours for determination of cell densities by spectrophotometric measurement. The quantification of the test item in the test samples was performed using high performance liquid chromatography (HPLC) with UV detection. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. At the start of the test 100 % of the nominal test concentrations were found (average of all test concentrations). After 72 hours test duration, 83 % of the nominal value was determined (average of all test concentrations). Intermediate samples at 24-hours and 48-hours were taken to track the concentration decrease during the course of the test. During the test the algae were exposed to a mean of 93 % of the nominal concentration. All reported results refer to nominal values. Thus, the 72-hour E_yC₅₀ was calculated to be > 93.9 mg test item/L and the 72-hour E_rC₅₀ value was calculated to be > 93.9 mg test item/L. The 72-hour NOE_yC was determined to be 30.3 mg test item/L and the associated 72-hour LOE_yC was 93.9 mg test item/L. The 72-hour NOE_rC was determined to be 30.3 mg test item/L and the associated 72-hour LOE_rC was 93.9 mg test item/L. No abnormal appearance of the algal cells was observed during the experiment.