Fatty acids, C8-10

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May - 11 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen

Analytical monitoring:

yes

Details on sampling:

- Sampling method: for assessing the concentration of the test item at test start, the samples were taken from the test solutions prior to the addition of the algae at test start. At test end the samples were taken from the pooled replicates per treatment level.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The preparation of the test media was performed under sterile conditions as far as possible. The highest test concentration was directly prepared by weighing 80 mg of the test item on a sterile teflon weighing boat. The weighing boat was given into a 1 L graduated glass flask and stirred strongly for 1 hour (with a sterile magnetic stir bar). The highest test concentration (conc. number 5) was diluted with 10-fold concentrated growth medium and ultra pure water under sterile conditions. Details are given below:

Conc. number Nominal conc. [mg/L] Test media/Conc. number 10-fold concentrated growth medium Ultra pure water
5 80.0 1000 mL no. 5 - -
4 40.0 500 mL no. 5 50 mL 450 mL
3 20.0 500 mL no. 4 50 mL 450 mL
2 10.0 500 mL no. 3 50 mL 450 mL
1 5.0 500 mL no. 2 50 mL 450 mL
Control 0 - 100 mL 900 mL

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: 61.81 SAG
- Source (laboratory, culture collection): SAG, Culture Collection of Algae at Pflanzenphysiologisches Institut of the University at Göttingen, Albrecht von Haller Institut, Untere Klarspüle 2, D-37073 Göttingen, Germany
- Method of cultivation: The stock cultures were maintained fulfilling the criteria of the OECD guideline No. 201

ACCLIMATION
- Acclimation period: Three days prior to testing a pre-culture was established in standard OECD growth medium to obtain exponentially-growing algae for the test
- Culturing media and conditions (same as test or not): yes

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

21 - 23 °C

pH:

7.71 (start) to 9.02 - 9.17 (end) (control)
6.40 - 7.73 (start) to 7.01 - 9.15 (end) (test cultures)

Nominal and measured concentrations:

control, 5, 10, 20, 40, and 80 mg/L (nominal)
control, 4.45, 9.07, 17.52, 35.10, and 71.00 mg/L (measured, initial)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): covered, with air-permeable silicone-sponge caps
- Material, size, headspace, fill volume: 250 mL conical glass flasks filled with 100 mL of the test media
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 10^4 cells/mL
- Control end cells density: 355 x 10^4
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8

GROWTH MEDIUM
- Standard medium used: yes/no
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: All stock solutions and the medium were prepared with purified water processed using an ELGA „PURELAB Ultra“.
- Culture medium different from test medium: no; sterilised synthetic OECD medium according to OECD 201 was used as growth medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH:
- Photoperiod: continuous
- Light intensity and quality: 7405 - 8147 Lux (OSRAM “day light”)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): cell concentrations were determined after 24, 48 and 72 h
- Determination of cell concentrations: electronic particle counter (CASY 1 Model TT, Roche Diagnostics GmbH, Germany). The correctness of the electronic counts was checked by microscopically counting following internal standard operation procedures

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: yes
- Test concentrations: 10, 50 and 100 mg/L and 2, 10 and 20 mg/L
- Results used to determine the conditions for the definitive study: Based on on the results in the range-finding study with 90.9% inhibition at a concentration of 100 mg/L and 5% inhibition at 10 mg/L, concentrations for the definitive study were chosen to be 5, 10, 20, 40, and 80 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

43.73 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 38.40 - 50.37

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

17.52 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

15.59 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 10.84 - 19.57

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

35.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

23.28 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 21.44 - 25.97

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

16.89 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CL: 15.04 - 18.38

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

17.52 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

35.1 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Results with reference substance (positive control):

EC50 (growth rate) = 2.91 mg/L (95% confidence limits 2.35 - 3.63 mg/L)

Reported statistics and error estimates:

The test results showing inhibitions around 50% were statistically analysed to determine an EC50, EC10 and EC10 values together with 95 % confidence intervals using Probit-analysis [Finney. D.J.: Statistical Method in Biological Assay. 2nd ed. London. 1984] assuming log-normal distribution of the values. Individual replicate responses were used for the regression analysis.
The NOEC and LOEC values for growth rate were determined by the Welch-t test for Inhomogeneous Variances with Bonferroni correction [Wilcoxon. Frank (1945): Individual Comparisons by Ranking Methods. Biometrics Bulletin 1: 80–83; Mann. Henry & Whitney. Donald (1947): On a test of whether one of two random variables is stochastically larger than the other. Annals of mathematical Statistics 18: 50-60] and for yield by the Williams Multiple Sequential t-test [Williams. D.A. (1971) A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics. 27. 103-117; Williams. D.A. (1972): The comparison of several dose levels with a zero dose control. Biometrics 28. 519-531].
The computer program ToxRat [ToxRat® Professional 2.10. ToxRat® Solutions GmbH. Naheweg 15. D-52477 Alsdorf] was used for statistical evaluations.

Chemical analysis:

At test start the measured concentrations of octanoic acid ranged between 87.6 and 90.7% of the nominal values. The concentrations decreased during the test period to levels between 84.3 and 94.0% of the measured initial values or 74.9 and 82.5% of nominal, respectively. Therefore, the test was evaluated using the initial measured octanoic acid concentrations.

Table: Measured concentrations of the test item in the test solutions

Nominal concentrations [µg test item/L]	Measured concentrations
	Day 0		Day 3
	[µg test item/L]	% nominal	[µg test item/L]	% nominal	% of day 0 measured
Control	0		0		84.3
5	4.45	88.9	3.75	74.9	85.3
10	9.07	90.7	7.74	77.4	90.0
20	17.5	87.6	15.76	78.8	94.0
40	35.1	87.8	33.00	82.5	90.8
80	71.0	88.8	64.48	80.6	84.3

Definitive test:

Table: Percent inhibition of growth rate and yield by the test item compared to controls after 72 h.

Test item nominal [mg/L]	Test item measured [mg/L]	% Inhibition of growth rate	% Inhibition of yield
Control	0.0	0.0	0
5	4.45	0.2	1.4
10	9.07	1.8	10.1
20	17.5	2.4	12.8
40	35.1	50.8	95.0
80	71.0	66	98.2

Table: Yield (cell number increase) dependent on initial measured concentration of test item and time.

Concentration [mg test item/L]	Control	4.45	9.07	17.52	35.10	71.00
24 h	8.260	7.588	7.556	6.171	5.680	2.536
	8.861	7.146	7.213	6.168	4.591	2.475
	8.910	6.900	7.400	5.683	4.948	2.835
	8.570	7.406	6.986	7.276	4.023	2.188
	7.668	-
	7.438
	9.040
	8.831
Mean:	8.447	7.260	7.289	6.325	4.811	2.508
Std.Dev.:	0.605	0.301	0.246	0.675	0.694	0.265
n:	8.000	4.000	4.000	4.000	4.000	4.000
CV:	7.200	4.100	3.400	10.700	14.400	10.600
48 h	55.840	51.890	54.230	40.670	11.960	4.548
	54.470	54.120	49.420	40.400	10.120	4.041
	56.650	46.220	55.300	36.850	9.890	3.986
	52.940	56.500	50.870	45.960	9.970	3.551
	54.430	-
	57.410
	64.110
	56.360
Mean:	56.526	52.182	52.455	40.970	10.485	4.031
Std.Dev.:	3.387	4.398	2.767	3.755	0.988	0.408
n:	8.000	4.000	4.000	4.000	4.000	4.000
CV:	6.000	8.400	5.300	9.200	9.400	10.100
72 h	348.500	340.500	352.500	301.200	28.030	8.330
	338.200	359.900	305.400	326.200	14.610	6.660
	377.000	338.100	288.200	267.000	13.710	5.930
	331.000	359.200	328.400	342.200	14.680	5.000
	352.300	-
	361.800
	379.400
	346.900
Mean:	354.387	349.425	318.625	309.150	17.757	6.480
Std.Dev.:	17.304	11.736	27.950	32.776	6.863	1.408
n:	8.000	4.000	4.000	4.000	4.000	4.000
CV:	4.900	3.400	8.800	10.600	38.600	21.700

(Mean: arithmetic mean; Std. Dev.: standard deviation; n: number of replicates; CV: coefficient of variation). Cell number at test start: 10 000 cells/mL.

Table: Growth rates (1/d) dependent on initial measured concentration of test item and time.

Concentration [mg test item/L]	Control	4.45	9.07	17.52	35.10	71.00
24 h	2.226	2.150	2.147	1.970	1.899	1.263
	2.289	2.098	2.106	1.970	1.721	1.246
	2.294	2.067	2.128	1.900	1.783	1.344
	2.259	2.129	2.078	2.113	1.614	1.159
	2.160	-
	2.133
	2.307
	2.286
Mean:	2.244	2.111	2.115	1.988	1.754	1.253
Std.Dev.:	0.066	0.037	0.030	0.090	0.119	0.076
n:	8.000	4.000	4.000	4.000	4.000	4.000
CV:	2.900	1.700	1.400	4.500	6.800	6.000
	2.244	2.111	2.115	1.988	1.754	1.253
48 h	2.020	1.984	2.006	1.865	1.281	0.857
	2.008	2.005	1.960	1.862	1.204	0.809
	2.027	1.927	2.015	1.817	1.194	0.803
	1.994	2.026	1.974	1.925	1.198	0.758
	2.008
	2.034
	2.088
	2.025
Mean:	2.020	1.984	2.006	1.865	1.281	0.857
Std.Dev.:	2.008	2.005	1.960	1.862	1.204	0.809
n:	2.027	1.927	2.015	1.817	1.194	0.803
CV:	1.994	2.026	1.974	1.925	1.198	0.758
	2.008
72 h	2.020	1.984	2.006	1.865	1.281	0.857
	2.008	2.005	1.960	1.862	1.204	0.809
	2.027	1.927	2.015	1.817	1.194	0.803
	1.994	2.026	1.974	1.925	1.198	0.758
	2.008
	2.034
	2.088
	2.025
Mean:	1.957	1.953	1.921	1.911	0.963	0.666
Std.Dev.:	0.016	0.011	0.029	0.036	0.107	0.062
n:	8.000	4.000	4.000	4.000	4.000	4.000
CV:	0.800	0.600	1.500	1.900	11.100	9.300
	1.957	1.953	1.921	1.911	0.963	0.666

(Mean: arithmetic mean; Std. Dev.: standard deviation; n: number of replicates; CV: coefficient of variation)

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 May - 27 May 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

calculation of results based on nominal values despite >20% deviation from measured values

GLP compliance:

yes

Remarks:

according to the Japanese GLP Standard

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Vapour pressure: 1 mm at 125 °C
- Water solubility: < 1 mg/L
- log Pow: 4.09

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 1.8, 3.2, 5.6, 10 and 18 mg/L (nominal)
- Sampling method: At study initiation: test solution for analytic was separately prepared, since the volume was > 5% of test water in vessels. After exposure: test solution was sampled directly from each test vessel.
- Sample storage conditions before analysis: Analysis was conducted immediately after sampling, no storage.

Vehicle:

yes

Details on test solutions:

- Method: The test substance was dissolved in vehicle, and this stock solution was diluted in dilution water and stirred for 1 hour.
- Controls: dilution water with/without vehicle
- Chemical name of vehicle: dimethylformamide
- Concentration of vehicle in test medium: 0.1 mL/L
- Evidence of undissolved material: not observed

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: alga
- Strain: ATCC22662
- Source: American Type Culture Collection
- Age of inoculum: within 72 hours
- Method of cultivation: on rotary shaker (100 rpm)

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.7 - 23.8 °C (23 ± 2 °C)

pH:

at the initiation: 7.6 - 7.8
at the end: 7.4 - 7.6

Nominal and measured concentrations:

nominal concentration: 1.8, 3.2, 5.6, 10, 18 mg/L
measured concentration: at test initiation: 1.8, 3.0, 5.9, 8.9, 16 mg/L
at test end (percent of nominal): 0.52 (29%), 1.2 (38%), 3.2 (57%), 6.0 (60%), 14 (78%) mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask with an air-permeable stopper
- Type: open
- Material, fill volume: glass, 100 mL
- Initial cells density: 10000 cells/mL
- Control end cells density: 2620000 ± 130000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: not stated
- Intervals of water quality measurement: pH (0 and 72 hours), temperature, light intensity and number of revolutions (0, 24, 48, 72 hours) were determined.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: not adjusted
- Photoperiod: continuously
- Light intensity and quality: 4300 - 5000 lux


EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter; 24, 48, 72 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: performed
- Test concentrations: 2.0 and 20 mg/L
- Results used to determine the conditions for the definitive study: 2.0 mg/L: growth inhibition was not observed, 20 mg/L: 93 % growth inhibition was observed

Reference substance (positive control):

yes

Remarks:

potassium dichromate (reagent grade)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 14 - 17 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 5.5 - 6.3 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EbC50: 0.44 mg/L

Reported statistics and error estimates:

Barlett, Probit and Dunnett test

Table 1: Cell density of Selenastrum capricornutum after treatment with decanoic acid

Loading Rate  (mg/L)	Areal Mean Measured Conc. (mg/L)	Cell Densities (cells/mL)
			0 Hour	24 Hour	48 Hour	72 Hour
Control	-	Mean	10000	65300	407000	2620000
		SD	0	5600	12000	130000
Solvent Control	-	Mean	1000	66900	411000	2550000
		SD	0	3200	14000	190000
1.8	2.12	Mean	1000	64000	392000	2630000
		SD	0	7200	8000	60000
3.2		Mean	1000	54000	321000	2090000
		SD	0	3400	23000	50000
5.6		Mean	1000	46800	239000	1260000
		SD	0	1700	10000	80000
10		Mean	1000	33000	128000	465000
		SD	0	1900	8000	18000
18		Mean	1000	23100	47000	99700
		SD	0	1200	3500	6800

Table 2: Growth inhibition of Selenastrum capricortum after treatment with decanoic acid

Nominal concentration (mg/L)	Inhibition (%) Biomass	Inhibition (%) Growth rate
Control	0	0
Solvent Control	1.7	1.5
1.8	0.6	-0.7
3.2	20.4	0.9
5.6	49.3	10.9
10	79.0	28.3
18	94.6	60.4

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please refer to the category justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

43.73 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 38.40 - 50.37

Remarks:

Source: CAS 124-07-2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

15.59 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 10.84 - 19.57

Remarks:

Source: CAS 124-07-2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 14 - 17 mg/L

Remarks:

Source: CAS 334-48-5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Source: CAS 334-48-5

Description of key information

ErC50 (72 h) = 15 mg/L (nominal, growth rate, OECD 201); RA CAS 334 -48 -5

NOErC (72 h) = 3.2 mg/L (nominal, growth rate, OECD 201); RA CAS 334 -48 -5

Key value for chemical safety assessment

EC50 for freshwater algae:

15 mg/L

EC10 or NOEC for freshwater algae:

3.2 mg/L

Additional information

Good quality read across data from the two main constituents (decanoic and octanoic acid) of the substance fatty acids, C8 -10 are used to assess the toxicity to algae.

The study with the read across substance octanoic acid (2012) was conducted according to OECD 201 and GLP. The test organism Pseudokirchneriella subcapitata was exposed to nominal test item concentrations of 0 (control), 5, 10, 20, 40, and 80 mg/L (measured: 0, 4.45, 9.07, 17.52, 35.10, and 71 mg/L) for 72 h. The test substance concentrations were monitored during the experiment by GC-MS analysis. The test solutions were sampled prior to the addition of the algae at test start of the experiment while at test end the samples were taken from the pooled replicates per treatment level. The recovery of the test item concentrations at test start ranged between 87.6 and 90.7% of the nominal values and decreased to levels between 74.9 and 82.5% of nominal, respectively. The test showed growth rate inhibitions which resulted in an EC50 (72 h) of 43.73 mg/L and an EC10 (72 h) of 15.59 mg/L based on the measured initial concentrations.

The study with the read across substance decanoic acid (1999) was conducted according to OECD 201 and GLP. The test organism Pseudokirchneriella subcapitata was exposed to 5 nominal test item concentrations of 0 (control), 1.8, 3.2, 5.6, 10, 18 mg/L for 72 h. The substance was dissolved in dimethylformamide and monitored during the experiment by GC-MS analysis. While recoveries of the test item concentrations were above 80% of nominal at the start of the experiment, they decreased to 29 – 78% of the nominal values at the end of the experiment. The test showed growth rate inhibitions and resulted to an ErC50 (72 h) of 15 mg/L and an NOEC (72 h) of 3.2 mg/L (nominal). Since these were the most sensitive effect values obtained from the read across data, they were used for further assessment of the fatty acids, C8 -10 substance.